Long-term sequelae are highly prevalent one year after hospitalization for severe COVID-19

Many coronavirus disease 2019 (Covid-19) survivors show symptoms months after acute illness. The aim of this work is to describe the clinical evolution of Covid-19, one year after discharge. We performed a prospective cohort study on 238 patients previously hospitalized for Covid-19 pneumonia in 2020 who already underwent clinical follow-up 4 months post-Covid-19. 200 consented to participate to a 12-months clinical assessment, including: pulmonary function tests with diffusing lung capacity for carbon monoxide (DLCO); post-traumatic stress (PTS) symptoms evaluation by the Impact of Event Scale (IES); motor function evaluation (by Short Physical Performance Battery and 2 min walking test); chest Computed Tomography (CT). After 366 [363–369] days, 79 patients (39.5%) reported at least one symptom. A DLCO < 80% was observed in 96 patients (49.0%). Severe DLCO impairment (< 60%) was reported in 20 patients (10.2%), related to extent of CT scan abnormalities. Some degree of motor impairment was observed in 25.8% of subjects. 37/200 patients (18.5%) showed moderate-to-severe PTS symptoms. In the time elapsed from 4 to 12 months after hospital discharge, motor function improves, while respiratory function does not, being accompanied by evidence of lung structural damage. Symptoms remain highly prevalent one year after acute illness.

www.nature.com/scientificreports/ post-recovery in up to 50% of Covid-19 survivors 7,13 . Alongside respiratory dysfunction, motor impairment is often reported post-Covid-19. When assessed with a 6 min walking test (6MWT), a vast proportion of survivors to Covid-19 walked a distance lower than expected for their age-adjusted predicted values 14 . Similar results have been reported with other tools, such as the Short Physical Performance Battery (SPPB) or the 2-min walk test (2MWT) 15 . Finally, the impact of Covid-19 on mental health should not be neglected: according to different studies, anxiety and/or depression may be reported in up to 30% of patients months after recovery 9,16 and posttraumatic symptoms are also quite common 7 . Whilst a number of reports have provided an initial account of the post-Covid-19 sequelae, how these clinical manifestations evolve over time is uncertain. We previously described the early psychological and functional sequelae of Covid-19 infection 4 months after hospital discharge in a prospective cohort of 238 patients 13 . In this prospective observational study, we describe the long-term functional impairment of respiratory, motor, psychological and cognitive function 12 months after SARS-CoV-2 infection and compare these to the symptomatic burden at 4 months follow-up, in an attempt to clarify reversibility of symptomatology.

Results
General features of the study population. The main characteristics of participants are listed in Table 1.
We also stratified patients according to different categories of radiological severity: -None to mild involvement: score ≤ 5: N = 146 (76.8%); -Moderate involvement: score > 5 and ≤ 10: N = 26 (13.7%); -Severe involvement > 10: N = 18 (9.5%). In eTables 3 and 4 we report the results of the univariate analysis of factors associated with impaired DLCO. As shown in Table 2, at logistic regression, female gender was strongly associated with a DLCO < 80% at one-year follow-up, independently of other factors, whereas arterial hypertension, chronic kidney disease and radiological involvement were associated with having a DLCO < 60% at the 12-month time-point. Physical performance evaluation. 2 patients refused to perform physical assessment. The SPPB test was altered in 37/198 (18.7%) subjects. All other patients (N = 161) underwent a 2MWT; 14 patients (7.1%) walked a distance lower than expected for age and gender. Thus, 51 patients (25.8%) had some degree of functional impairment. None of them reported a new diagnosis of inflammatory arthritis or myopathy. This proportion is significantly lower than that reported in the same group of patients 4 months after hospital discharge (N = 103, 52.8%; − 27% CI95 (− 33; − 20%); p < 0.001).

Pulmonary function tests.
In eTable 5 we report the results of univariate analysis. At logistic regression (Table 3), DLCO and COPD were independently associated with motor impairment.
Thirty patients reported fatigue, although the number raised to 66 (33.3%), when this symptom was assessed by the CFS. A CFS consistent with fatigue was associated to: female gender (p = 0.0008); a higher BMI (

Discussion
Since the first outbreak of SARS-Cov-2 in Wuhan great emphasis has been devoted to the understanding of determinants of acute morbidity and mortality from Covid-19. More recent studies have highlighted the long-term consequences from SARS-Cov-2 infection in patients who survive severe disease, however data on long-term impact and reversibility of Covid-19-related sequelae are lacking. This is one of the first studies assessing the persistence of symptoms from severe Covid-19 one year after hospital discharge; according to our data, symptoms may persist in approximately 40% of patients. In a very recent report by Lombardo et al., the proportion of patients still complaining symptoms after 12 months is even higher (81%) 17 . In a large Chinese prospective cohort, Huang et al. reported that 76% of patients complained of at least one residual symptom 6 months after acute illness 9 , although lower rates (around 40-50%) have been reported by other authors 7,18,19 . These observations are clues that Covid-19 may cause persistent organ damage. However, symptoms are, by definition, subjective and might be influenced by psychological factors. This might also explain why some symptoms, such as cough and arthromyalgia were even more common at 12 than at 4 months. It does not probably reflect a progressive worsening of long Covid, rather suggesting a deeper insight of the general population about this issue. Indeed, the second time point visit of our study was carried out while the media were already informing the population about the longstanding consequences of SARS-CoV-2 infection, which were less known when we carried out the first time point visit. Thus, these differences are probably due to the fact that patients paid more attention to their symptoms.
Covid-19 pandemic has had a strong detrimental impact on the mental health of general population; according to a large international cohort of almost 10,000 subjects, the pandemic was experienced as at least moderately stressful for most people while 11% reported the highest levels of stress. Moreover, symptoms of depression were common; lack of social support, low education level, and precarious financial status were all predictors of worse psychological outcomes 20 . Restrictions to free movement and social distancing makes it difficult to estimate the background psychological impact of Covid-19 at population level. According to our data, both fatigue and dyspnea are associated to the IES score, suggesting that these physical symptoms and the mental health trauma derived from Covid-19 may have a role in the persistence of these symptoms. This is important, if we consider that in our cohort, the prevalence of subjects experiencing moderate to severe PTS symptoms did not significantly decrease over time, remaining stable from 4 to 12 months after discharge. Our data are in line with those of other authors suggesting a prevalence of PTS symptoms around 20% in patients recovered from Covid-19 21,22 and reflects, at least in part, the high level of stress in the population. Indeed, accordingly to a recent meta-analysis, the prevalence of PTS disorder in the general population during the pandemic is around 15% 23 .
In addition, impaired alveolar gas transfer assessed by DLCO observed in the first months following SARS-CoV-2 infection, did not show any appreciable improvement over time. This objective functional alteration is related to an anatomical damage 24 . Indeed, the CT severity score is a major determinant particularly for severe DLCO reduction, as reported previously 25 . This suggests that a structural damage subtends the development of respiratory impairment and is involved in the pathogenesis of dyspnea. However, as previously mentioned, the observation that the proportion of patients complaining dyspnea increased significantly over time, despite a stable DLCO, makes evident that this symptom has a relevant psychological background, as further confirmed by its association with IES. Therefore, we can summarize that beside patients with respiratory impairment, others may complain dyspnea mainly on a psychological basis.
Moreover, consistently with our previous findings 13 , DLCO reduction is strongly associated to motor impairment, suggesting a central role for respiratory system in driving physical functional limitations. Interestingly,  www.nature.com/scientificreports/ www.nature.com/scientificreports/ althogh DLCO did not improve over time, we observed a significant reduction of patients with motor limitations from 4 to 12 months. The improvement in motor function might reflect a beneficial effect of physical activity on around half of the patients; however, this aspect was not directly investigated and future research should help clarify whether exercise might improve long-term outcome from Covid-19.
Finally, our paper contributes to profiling the patients at higher risk for long-lasting sequelae. Different authors already reported that females are at higher risk for persistent symptoms and psychological involvement 8,9,26 , leading them to postulate a potential role for hormonal factors in the pathogenesis of this condition 27 . Our data are in line with the current literature, showing that female gender was associated to DLCO reduction, persistent dyspnea and fatigue. However, males have a poorer prognosis during acute illness 28 ; hence, females may survive more frequently to severe Covid-19, possibly justifying the persistence of clinical sequelae. This is a possible explanation, although probably not completely satisfactory; indeed, when included in a multivariate model, parameters assessing acute disease severity were not related to DLCO reduction. Conversely, some specific comorbidities are associated to persistent functional impairment, such as COPD, CKD and arterial hypertension, which is a well-described negative prognostic factor during the acute phase 29 .
The main limitation of our study is the unavailability of detailed clinical data (particularly PFTs) predating SARS-CoV-2 infection, preventing us to discriminate the damage attributable to Covid-19 from that pre-existing this disease. Although we demonstrated that DLCO reduction was associated to the extent of radiological damage, the absence of CT scans before the infection did not allow to estimate whether the structural damage is consistent with the evolution of the acute illness or the result of respiratory comorbidities. A second limitation is the relatively small sample size; and a drop-out rate of 16% compared to the 4 month follow up timepoint. This was expected, also considering that the current follow-up visit coincided with an outbreak of the pandemic in March-April 2021. Despite the acknowledged limitations, the percentage of patients attending the one-year follow-up visit was > 80%, being representative of the original cohort.
In conclusion, symptoms of Covid-19 may persist up to 12 months after hospital discharge as a consequence of both mental stress and organ damage. Altered respiratory function secondary to radiologically proven structural lung damage and mental health sequelae persist over time in a significant proportion of patients. Conversely, the recovery of motor function continues during the follow-up from 4 to 12 months. Female gender, arterial hypertension, chronic kidney disease and COPD may represent risk factors for persistent Covid-19 sequelae.

Methods
Study population. Briefly, as described in the previously published paper 13 , we contacted all the 767 patients (or their caregivers) aged 18 years or older who were discharged between March 1 and June 29, 2020, from the Azienda Ospedaliero-Universitaria Maggiore della Carità university hospital in Novara, Italy, Northern Italy, where they had been admitted for a confirmed diagnosis of Covid-19. Out of them 238 consented to participate to a follow-up visit performed 3 to 4 months after discharge; we contacted them again to schedule a further visit after 12 months. Patient disposition is reported in Fig. 1.
One patient was excluded because she was pregnant, while 6 more patients were lost to the follow-up. Thirtyone declined participation to the second time-point of the study (see Fig. 1 for further details) mainly because of impossibility to attend the visit (health or work issues) and/or fears of new in-hospital exposure to SARS-CoV-2. The eligible population included a total 200 patients, 84% of the originally recruited cohort, who were hospitalized between 1st March 2020 and 15th May 2020.
All patients were contacted telephonically and offered a clinical evaluation 12 months after hospital discharge: the visits were scheduled between 15th March and 4th May 2021.
All participants signed an informed consent and underwent a Complete clinical assessment including physical examination to investigate: -Severity of acute illness, classified using an eight-category scale, as previously published 30 ; -Symptoms at SARS-CoV-2 diagnosis and at the 4-months evaluation; -Co-morbidities, home medications and cumulative illness rating scale (CIRS) 31 ; The clinical evaluation was performed by hospital specialists involved in the management of Covid-19 patients and data recorded in an electronic case report form.
The study protocol was approved by the local ethical committee: Comitato Etico Interaziendale Novara (IRB code CE 117/20) and conducted in strict accordance with the principles of the Declaration of Helsinki. All the subjects who participate to the study signed a written informed consent.
The present report follows STROBE guidelines for cohort studies.
Pulmonary function tests. All patients underwent standard pulmonary function testing (PFT) (with a Quark PFT with X9 pneumotach, COSMED srl, Roma, Italy) for forced expiratory volume in one second (FEV1), vital capacity (VC), forced vital capacity (FVC), DLCO and total lung capacity (TLC). DLCO and TLC were determined by the single-breath carbon monoxide technique. The examination was performed on the same day of the visit.
Physical performance tests. On the same day, we also assessed the patient's physical performance; patients underwent a Short Physical Performance Battery (SPPB) as previously described: the score is considered as pathological when 10 or lower 34,35 . For those patients with SPPB score > 10 we also performed a 2-min walk test (2MWT), which was compared with reference data values for an age-matched population, aiming to disclose more subtle functional alterations 36-38 . Mental health symptoms tests. We assessed the prevalence of post-traumatic stress (PTS) symptoms by administering the Impact of Event Scale (IES) 39 . A total score between 0 and 8 is interpreted as subclinical PTS; 9-25 as mild PTS; 26-43 as moderate PTS and > 44 as severe PTS. Internal consistency coefficients for intrusion is 0.84, for avoidance is 0.71 40 .
Radiological assessment. Chest computed tomography (CT) scans were performed during single full inspiratory breath hold in supine position on a 256-slice CT (PHILIPS Brilliance ICT). All images were reconstructed with a slice thickness of 1 mm.
The images in DICOM (Digital Imaging and COmmunications in Medicine) extension files were transferred to the Picture Archiving and Communication System (PACS) and then analyzed into a workstation equipped with two 35 × 43 cm monitors (produced by Eizo, with 2048 × 1536 matrix). www.nature.com/scientificreports/ All CT scans have been evaluated by a radiologist with more than 10 years of experience in chest CT scan, blinded to clinical data. Both lung (width, 1600 HU; level, − 550 HU) and mediastinal (width, 400 HU; level, 40 HU) window settings were evaluated.
Statistical analysis. Data were analysed using the statistical software Stata 15.1 (StataCorp, College Station, USA). Normality was assessed by Shapiro-Wilk test. The measures of centrality and dispersion for continuous variables were medians and [25th-75th percentile]; comparisons between groups for these variables were performed by the Mann-Whitney test. The median values of respiratory functional parameter were compared by Wilcoxon's test for paired samples; the prevalence of DLCO reduction below the thresholds considered and the prevalence of physical impairment between 4 and 12 months were compared by McNemar's test. Categorical variables, whenever dichotomous or nominal, were reported as frequencies and percentages, and analyzed through the Pearson's chi-square or Fisher's exact test, as appropriate. The primary endpoint was the proportion of patients with a DLCO < 80% of predicted value. The original cohort was sufficiently powered to detect as statistically significant a 0.12 increase in the proportion of patients with DLCO < 80% of predicted among Covid-19 survivors compared to that observed in a reference population (0.30 vs. 0.18, respectively), with an alpha level = 0.005. Secondary endpoints were: proportion of subjects showing severe DLCO (< 60%) impairment, potentially associated to a higher risk of pulmonary fibrosis; prevalence of motor impairment (SBBP score < 11 or SBBP score ≥ 11 and altered 2MWT); prevalence of moderate-to-severe PTS symptoms (IES > 26 and CDPI > 28); factors associated to the previous endpoints.
To disclose associations, we run a univariate analysis, including: the comorbidities with a biological rationale and CIRS, age, gender, smoking attitude, intensive care unit (ICU) admission during hospital in-stay, length of hospital in-stay, modality of oxygen delivery during hospital in-stay, class severity of acute illness, CT severity score. All variables associated with the outcome of interest at a p-value threshold below 0.20 were included in logistic regression models. www.nature.com/scientificreports/ Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/.