Pulmonary long-term consequences of COVID-19 infections after hospital discharge

Journal Pre-proof Pulmonary long-term consequences of COVID-19 infections after hospital discharge J.R. Blanco, M.J. Cobos-Ceballos, F. Navarro, I. Sanjoaquin, F. Arnaiz de las Revillas, E. Bernal, L. Buzon-Martin, M. Viribay, L. Romero, S. Espejo-Pérez, B. Valencia, D. Ibañez, D. Ferrer-Pargada, D. Malia, F.G. Gutierrez-Herrero, J. Olalla, B. Jurado-Gamez, J. Ugedo PII: S1198-743X(21)00101-4 DOI: https://doi.org/10.1016/j.cmi.2021.02.019 Reference: CMI 2434 To appear in: Clinical Microbiology and Infection Received Date: 11 November 2020 Revised Date: 9 February 2021 Accepted Date: 18 February 2021 Please cite this article as: Blanco J, Cobos-Ceballos M, Navarro F, Sanjoaquin I, Arnaiz de las Revillas F, Bernal E, Buzon-Martin L, Viribay M, Romero L, Espejo-Pérez S, Valencia B, Ibañez D, FerrerPargada D, Malia D, Gutierrez-Herrero F, Olalla J, Jurado-Gamez B, Ugedo J, Pulmonary long-term consequences of COVID-19 infections after hospital discharge, Clinical Microbiology and Infection, https://doi.org/10.1016/j.cmi.2021.02.019. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2021 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved. Title: Pulmonary long-term consequences of COVID-19 infections after hospital discharge AUTHORS: JR. Blanco 1,2 MJ. Cobos-Ceballos 3,4 F. Navarro 5 I. Sanjoaquin 6 F. Arnaiz de las Revillas 7 E. Bernal 8 L. Buzon-Martin 9 M. Viribay 10 L. Romero 2 S. Espejo-Pérez 3,11 B. Valencia 12 D. Ibañez 13 D. Ferrer-Pargada 14 D. Malia 15 FG. Gutierrez-Herrero 16 J. Olalla 5 B. Jurado-Gamez 3,4 J. Ugedo 17 Journal Pre-proof 1. Servicio de Enfermedades Infecciosas. Hospital Universitario San Pedro, Logroño, La Rioja, SPAIN. 2. Centro de Investigación Biomédica de La Rioja, Logroño, La Rioja, SPAIN 3. Instituto Maimónides de Investigación Biomédica de Córdoba. Universidad de Córdoba. SPAIN 4. Servicio de Neumología. Hospital Universitario Reina Sofía, Córdoba. SPAIN 5. Servicio de Medicina Interna. Hospital Costal de Sol, Marbella. Málaga. SPAIN 6. Servicio de Enfermedades Infecciosas. HCU Lozano Blesa, Zaragoza. SPAIN 7. Servicio de Enfermedades Infecciosas. H Universitario Marqués de Valdecilla, Santander. SPAIN 8. Sección de Enfermedades Infecciosas. Hospital General Universitario Reina Sofía de Murcia. Universidad de Murcia. SPAIN 9. Servicio de Medicina Interna. Hospital Universitario de Burgos. SPAIN 10. Vitro, Labora 11. Servicio de Radiología. Hospital Universitario Reina Sofía, Córdoba. SPAIN 12. Servicio de Neumología. Hospital Costal de Sol, Marbella. Málaga. SPAIN 13. Servicio de Radiología. HCU Lozano Blesa, Zaragoza. SPAIN 14. Servicio de Neumología. H Universitario Marqués de Valdecilla, Santander. SPAIN 15. Servicio de Neumología. Hospital General Universitario Reina Sofía de Murcia. SPAIN 16. Servicio de Neumología. Hospital Universitario de Burgos. SPAIN 17. Servicio de Neumología. Hospital Universitario San Pedro, Logroño, La Rioja. SPAIN Keywords: COVID-19, Intracellular adhesion molecule, Lung diffusion capacity, Osteoprotegerin, Plasminogen activator inhibitor, Tissue inhibitor of matrix metalloproteinases, Tomography Corresponding author: Dr. José-Ramón Blanco Hospital San Pedro – Centro de Investigación Biomédica de La Rioja (CIBIR) Departamento de Enfermedades Infecciosas Piqueras 98, 26006 Logroño, La Rioja, Spain Telephone: +34 941298993 Email: [email protected] ; [email protected] Journal Pre-proof ABSTRACT Objectives: COVID-19 survivors are reporting residual abnormalities after discharge from the hospital. Limited information is available about this stage of recovery or the lingering effects of the virus on pulmonary function and inflammation. The aim of this study was to describe lung function and to identify biomarkers in serum and induced sputum samples from patients recovering from COVID-19 hospitalisation. Methods: Patients admitted to Spanish hospitals with laboratory-confirmed COVID-19 infection by a real-time PCR (RT-PCR) assay for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) were recruited for this study. Each hospital screened their lists of discharged patients at least 45 days after symptom onset. SARS-CoV-2-infected patients were divided into mild/moderate and severe disease groups according to the severity of their symptoms during hospitalisation. Patients’ epidemiological and medical histories, comorbidities, chronic treatments, and laboratory parameters were evaluated. Pulmonary function tests, the standardised 6-minute walk test (6 MWT) and chest computed tomography (CT) were also performed. The levels of proteases, their inhibitors, and shed receptors were measured in serum and induced sputum samples. Results: A total of 100 patients with respiratory function tests were included in this study. The median number of days after the onset of symptoms was 104 (IQR 89.25, 126.75). COVID-19 was severe in 47% (47/100) of patients. CT was normal in 48% (48/100) of patients. Lung function was normal (FEV1 ≥80%, FVC ≥80%, FEV1/FVC ≥0.7, and diffusing capacity for carbon monoxide [DLCO] ≥80%) in 92% (92/100), 94% (94/100), 100% (100/100) and 48% (48/100) of patients, respectively. Multivariate analysis showed that a DLCO <80% (OR 5.92; 95%CI 2.28-15.37; p <0.0001) and a lower serum LDH level (OR 0.98; 95%CI 0.97-0.99) were associated with the severe disease group of SARS-CoV-2 during hospital stay. Conclusions: A diffusion deficit (DLCO <80%) was still present after hospital discharge and was associated with the most severe SARS-CoV-2 cases. Journal Pre-proof INTRODUCTION Approximately 104 million individuals worldwide have recovered from COVID-19 (https://coronavirus.jhu.edu/map.html). However, some survivors report persistent severe symptoms and organ dysfunction [1]. These symptoms might be, in part, a consequence of the cytokine storm suffered in the acute phase of the infection [2]. Previous studies have shown that higher levels of proinflammatory cytokine responses during the acute phase of other coronavirus infections such as severe acute respiratory syndrome (SARS) [3] and the Middle East Respiratory Syndrome Coronavirus (MERS-CoV) [4], were associated with severe lung disease. Unlike previous coronaviruses, COVID-19 does not seem to be just a respiratory affliction; rather, it is a viral infectious process involving multiple systems [5]. Residual lung abnormalities have been found in patients with SARS-CoV-2 1-3 months after discharge from the hospital [6-9]. However, limited information is available about the serum inflammatory state during recovery from SARS-CoV2. The impact of residual inflammation on the lungs is even rarer. Because the persistence of this inflammatory state in blood and sputum could have important prognostic implications, we performed this study. METHODS Participants This was a prospective study of patients older than 18 years of age who were admitted to different Spanish hospitals with laboratory-confirmed COVID-19 infection by real-time PCR (RT-PCR) assay for SARS-CoV-2. Each hospital screened their lists of discharged patients. These patients were interviewed by phone at least 45 days after symptom onset and asked to collaborate if they met inclusion criteria. Exclusion criteria included patients with a need for prior invasive mechanical ventilation, chronic infectious diseases, chronic lung diseases, concurrent autoimmune or cancer diseases, chronic use of corticosteroids or immunosuppressive therapy, pregnancy, alcohol/drug abuse, or patients whose conditions did not allow participation in this study. The study was approved by the Institutional Research Ethics Committees. All participants provided written informed consent. Journal Pre-proof Patients were divided into mild (mild and moderate) and severe groups according to the severity of their symptoms during their hospital stays. The mild group did not have pneumonia imaging; the moderate group showed pneumonia; and the severe group had dyspnoea, respiratory frequency ≥ 30/minute, blood oxygen saturation ≤93%, PaO 2 /FiO 2 ratio <300, and/or lung infiltrates >50% of the lung field within 24-48 hours [10]. Patients requiring invasive mechanical ventilation were excluded because of its impact on systemic inflammation [11]. Epidemiological, medical history, comorbidities, chronic treatments, and laboratory parameters were evaluated. Smoking status was determined from self-administered survey responses. Anthropometric measurements included body mass index (BMI). At least 45 days after symptom onset, pulmonary function testing, standardised 6-minute walk tests (6 MWT) [12], and chest computed tomography (CT) were performed. Lung function included forced vital capacity (FVC), forced expiratory volume in the first second (FEV1), FEV1/FVC ratio and diffusion capacity of the lung for carbon monoxide (DLCO). Diffusion deficit was considered a DLCO <80% of the predicted value [13]. The 6 MWT, a practical and simple test that provides a global measure of functional capacity, was performed in accordance with international recommendations [12]. Peripheral oxygen saturation (SpO 2 ) was monitored using a handheld oximeter. ∆SpO 2 -6 MWT was defined as the difference between the resting and nadir SpO 2 . The 6 MWT distance was also evaluated [14]. CT was considered normal in the absence of ground-glass opacification, crazy-paving patterns, consolidation, or linear opacities [15]. The levels of biomarkers were measured in serum and induced sputum samples. Serum samples were obtained from blood drawn at a date close to the tests already described and stored at -80ºC. Sputum was induced as previously described [16] and stored at -80°C. It was obtained, whenever possible, on the same day as the respiratory function tests. The concentrations of multiple proteases and their inhibitors (plasminogen activator inhibitor [PAI]-1, PAI-2, and tissue inhibitor of matrix metalloproteinases [TIMP]-1). Shed receptor (intracellular adhesion molecule [ICAM]-1, ICAM-3, osteoprotegerin [OPG]) Journal Pre-proof were evaluated in serum and sputum samples. These parameters were analysed in duplicate employing commercially available ELISA kits. The lower detection limits are shown (Supplementary Table 1). All samples were tested individually (one sample per well), but samples from all groups were measured on the same plate. The hook effect, a state of antigen excess relative to the antibody probes, resulting in falsely lowered values, was ruled out after analysing undiluted and diluted samples. Data analysis Categorical variables were reported as frequencies and proportions. Continuous variables with a normal distribution are presented as the mean (standard deviation [SD]), and those with a non-normal distribution are presented as the median (interquartile range values [IQR] p25, p75). To compare the demographic and clinical variables between groups, the chi-square test or Fisher's exact test was used for each categorical variable, as appropriate. For quantitative variables, the nonparametric Mann-Whitney U test was used. Multivariate analysis was carried out using binary logistic regression with the forward conditional method, introducing DLCO (<80 vs. ≥80%) as the dependent variable. Independent variables were all variables that were statistically significant in the bivariate analysis, or clinical implications. The results of the multivariate model were adjusted, and we present the odds ratio and its 95% confidence interval (CI). Statistical significance was set at p <0.05. Analyses were performed using SPSS 24.0 software (SPSS Inc., Chicago, IL, USA). RESULTS A total of 108 patients were included in this study. Of the sample, 100 had adequate respiratory function tests. Most (69%9 were >50 years (69/100), 64% were male (64/100), and 90% were Caucasian (90/100). The median number of days after the onset of symptoms was 104 (IQR 89.25, 126.75). Common comorbidities included hypertension (25%; 25/100), diabetes mellitus (10%; 10/100), cardiovascular disease (4%; 4/100), and chronic kidney disease (2%; 2/100). Obesity (BMI ≥30%) was present in 37% (37/100), and 59% never had smoked (59/100). Chronic therapy included angiotensin-converting enzyme Journal Pre-proof inhibitors/angiotensin II receptor blockers use (17%, 17/100), statin use (12%, 12/100), and aspirin use (3%, 3/100). COVID-19 was severe in 47% of patients (47/100). Lung function was normal (FVC ≥80%, FEV1 ≥80%, FVC/FEV1 ≥0.7, and DLCO ≥80%) in 92% (92/100), 94% (94/100), 100% (100/100), and 48% (48/100), respectively. Control CT was normal in 48% (48/100). Given the high percentage of subjects with DLCO <80% and its involvement in lung damage, this lung parameter was evaluated. Table 1 shows the patient characteristics according to DLCO severity. With the exception of significant data referring to the severity of COVID-19 disease during hospitalisation and the length of hospital, no other significant differences were observed. Table 2 shows the analytical parameters according to DLCO severity. Finally, Table 3 provides information about the tests carried out and the minimum time elapsed until tests were performed. No differences were observed after analysing ∆SpO 2 -6 MWT (data not shown). Multivariate analysis showed that a DLCO <80% was associated with severe disease in the SARS-CoV-2 group during their hospital stays (OR 5.92; 95% CI 2.28-15.37; p <0.0001) as were lower serum LDH levels (OR 0.98; 95% CI 0.97-0.99; p 0.002). DISCUSSION Since the SARS-CoV-2 outbreak, there has been increasing concern about the potential risk of parenchymal fibrosis and lung function impairment. The most important factor is lung diffusion capacity [17]. Zhao et al. [6] reported that three months after COVID-19 discharge, a high percentage of CT abnormalities (70.9%) and DLCO anomalies (16.4%) were still present in recovering patients. Other authors, such as Mo et al. [7], reported that nearly a month after hospital discharge that, regardless of the degree of SARS-CoV-2 severity, no significant differences in FEV1, FVC, or its ratio were observed. However, the DLCO value was significantly lower as the severity of the clinical picture increased (47.2% in total; 30.4% in mild illness and 84.2% in severe pneumoniae). In this study, the authors included a small number of patients with previous pulmonary pathology, Journal Pre-proof one of the exclusion criteria of our study. Likewise, Frija-Masson et al. [9] also observed that more than half of patients with COVID-19 pneumonia, some of whom had respiratory comorbidities, exhibited abnormal lung function one month after symptom onset, without a clear relationship with pneumonia extent on chest CT. Huang et al. [18] observed that 30 days after discharge from the hospital, patients exhibited nearly significant differences in DLCO values (<80%), 42.5% in non-severe cases, and 75.6% in severe cases (p < 0.053). In that study, patients with a previous history of pulmonary resection, neurological disease, or mental illness were excluded. In our study, DLCO findings were close to those observed by Mo et al. [7] and Huang et al. [18], while the CT findings were clearly better than those reported by Zhao et al. [6]. After the 2003 outbreak of SARS, survivors evaluated within three months of discharge showed that lung fibrotic changes occurred mostly in severely sick patients [19]. These same authors also observed that when assessing lung fibrotic changes, DLCO scores were more sensitive than chest radiography and/or high-resolution CT. These results are similar to those observed by our group. During the follow-up of SARS patients, abnormal CT (30%) and impaired DLCO function (15.5%) [20] were still present six months later. These authors also observed significant impairment in DLCO function (23.7%) one year after illness onset [21]. All these data suggest that some of the recovered COVID-19 patients will have significantly impaired lung function months after discharge. Contrary to our expectations, LDH levels were significantly lower in patients with DLCO abnormalities (<80%). However, serum LDH is a sensitive, burdensome marker for cell injury [22]. One of the reasons could be that its levels vary in multiple circumstances (cell damage related to ischaemia, exposure to bacterial toxins, chemical poisoning, etc.), which is why serum LDH is difficult to use as a valid biomarker of lung damage or inflammation [22]. However, some authors have observed that LDH (cut-off value of 344.5 U/L) could be a predictive factor for early recognition of lung injury and severe COVID-19 cases [23]. These levels are clearly higher than those presented by our patients. Journal Pre-proof [32] Goshua G, Pine AB, Meizlish ML, Chang CH, Zhang H, Bahel P, et al. Endotheliopathy in COVID-19-associated coagulopathy: evidence from a singlecentre, cross-sectional study. Lancet Haematol. 2020;7(8):e575-e582. [33] Ackermann M, Verleden SE, Kuehnel M, Haverich A, Welte T, Laenger F, et al. Pulmonary vascular endothelialitis, thrombosis, and angiogenesis in Covid-19. N Engl J Med. 2020;383(2):120-128. [34] Tsoutsou PG, Gourgoulianis KI, Petinaki E, Mpaka M, Efremidou S, Maniatis A, et al. ICAM-1, ICAM-2 and ICAM-3 in the sera of patients with idiopathic pulmonary fibrosis. Inflammation. 2004;28(6):359-364. [35] Taz TA, Ahmed K, Paul BK, Kawsar M, Aktar N, Mahmud SMH, et al. Network-based identification genetic effect of SARS-CoV-2 infections to Idiopathic pulmonary fibrosis (IPF) patients. Brief Bioinform. 2020. doi: 10.1093/bib/bbaa235. [36] Liu Y, Yan LM, Wan L, Xiang TX, Le A, Liu JM, et al. Viral dynamics in mild and severe cases of COVID-19. Lancet Infect Dis. 2020;20(6):656-657. Journal Pre-proof Table 1. Patient characteristics among SARS-CoV-2 survivors according to DLCO severity. DLCO <80 (n = 52) DLCO ≥80 (n = 48) P value Age in years, mean (± SD) Age >50 years, n (%) 54.98 ±10.72 34 (65.4) 54.75 ± 9.83 35 (72.9) 0.911 0.416 Male sex, n (%) 33 (63.5) 31 (64.6) 0.907 Caucasian, n (%) 47 (90.4) 43 (89.6) 0.894 Never smoker history, n (%) 32 (61.5) 27 (56.3) 0.591 Comorbidities Cardiovascular disease, n (%) 4 (7.7) 0 (0) 0.119 Hypertension, n (%) 15 (28.8) 10 (20.8) 0.355 Diabetes mellitus, n (%) 7 (13.5) 3 (6.4) 0.324 Chronic renal failure, n (%) 2 (3.8) 0 (0) 0.496 Chronic aspirin use, n (%) 2 (3.8) 1 (2.1) 1.000 Chronic statin use, n (%) 8 (15.4) 4 (8.3) 0.362 Chronic ACE/ARA-II use, n (%) 9 (17.3) 8 (16.7) 0.932 SARS - CoV - 2 data during hospital ization admission Severity disease during hospital admission, n (%) 34 (65.4) 13 (27.1) <0.0001 Days of hospitalization , median (p25, p75) 7.0 (5.0; 9.75) 8.0 (6.0; 11.0) 0.038 Note: ACE = angiotensin converting enzyme inhibitors; ARA-II = angiotensin II receptor blockers; BMI = Body mass index; DLCO = diffusion capacity of the lung for carbon monoxide; SD = Standard deviation Journal Pre-proof Table 2. Analytical characteristics among SARS-CoV-2 survivors according to DLCO severity. DLCO <80 (n = 52) DLCO ≥80 (n = 48) P value Serum parameters , median (p25, p75) WBC count, cells/μL 6.10 (5.30; 6.59) 5.70 (5.0; 6.6) 0.383 Glucose, mg/dL 97.0 (93.25; 112.7) 32.4 (27.7; 39.9) 0.016 Creatinine, mg/dL 0.87 (0.74; 1.01) 0.87 (0.76; 0.98) 0.970 ALT, UI/L 21.0 (16-0; 32.0) 24.0 (18.0; 33.0) 0.224 AST, UI/L 22.0 (17.0; 25.0) 24.0 (20.0; 27.0) 0.034 LDH , UI/L 187.0 (164.0; 201.0) 196.0 (174.2; 256.7) 0.006 CRP g/dL 3.0 (1.0; 4.0) 4.0 (1.0; 4.0) 0.751 OPG pg/ml 62.6 (48.0; 81.0) 58.0 (48.4; 72.5) 0.410 TIMP-1 ng/ml 278.1 (249.8; 306.8) 281.3 (242.6; 312.3) 0.598 ICAM-1 ng/ml 169.9 (131.5; 245.9) 173.5 (122.5; 243.6) 0.738 ICAM-3 ng/ml 141.8 (115.8; 187.9) 136.6 (106-2; 163.0) 0.143 PAI-1 ng/ml 125.6 (98.2; 146.5) 119.2 (107.3; 142.4) 0.945 PAI-2 ng/ml 4.3 ±(2.8; 6.7) 3.7 (2.1; 5.2) 0.492 Induced sputum samples , median (p25, p75) OPG pg/ml 1.0 (1.0; 9.71) 1.0 (1.0; 11.1) 0.912 TIMP-1 ng/ml 68.9 (35.0; 120.4) 48.5 (35.0; 75.1) 0.112 ICAM-1 ng/ml 2.2 (0.9; 3.5) 2.21 (0.1; 2.38) 0.083 ICAM-3 ng/ml 34.2 (12.6; 86.1) 38.27 (16.2; 92.4) 0.812 Note: ALT = Alanine aminotransferase; AST = Aspartate aminotransferase; CRP = C-reactive protein; DLCO = diffusion capacity of the lung for carbon monoxide; ICAM = Intracellular adhesion molecule; LDH = Lactate dehydrogenase; OPG = Osteoprotegerin; PAI = Plasminogen activator inhibitor; TIMP = Tissue inhibitor of matrix metalloproteinase; WBC = White blood cell count . Journal Pre-proof Table 3. Pulmonary function test and computed tomography among SARS-CoV-2 survivors according to DLCO severity. DLCO <80 (n = 52) DLCO ≥80 (n = 48) P value Days after symptoms onset * Days after symptoms onset >90, n (%) 100.0 (87.5; 108.7) 36 (69.2) 11 4 . 5 (94.2; 133.7) 38 (79.2) 0.012 0.258 Functiona l lung parameter and imaging CT FVC (%)* FVC >80%, n(%) 106.9 (91.0; 113.7) 48 (92.3%) 104.5 (94.7; 114.7) 46 (95.8%) 0.904 0.906 FEV1 (%)* FEV1 >80%, n (%) 102.5 (94.1; 113.0) 48 (91.7) 107.2 (98.0; 118.0) 44 (91.7) 0.214 0.458 FEV1/FVC ratio* 1.0 (0.9; 1.0) 0.97 (0.92; 1.01) 0.066 6MWT distance, mean (± SD) 6MWT distance >550,n (%) 5 13 . 0 (450.0; 594.6) 20 (39.2) 577.0 (540.0; 645.0) 31 (66) 0.001 0.008 Pathologic CT, n (%) 31 (59.6) 20 (42.6) 0.900 Note: Data presented as median (P25; P75)*; 6MWT = 6-minute walk test; CT = chestcomputed tomography; DLCO = diffusion capacity of the lung for carbon monoxide; FEV1 = forced expiratory volume in the first second; FVC = forced vital capacity; SD = Standard deviation Journal Pre-proof