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. 2023 Feb 16;18(2):e0281249. doi: 10.1371/journal.pone.0281249

Association between serum ferritin level and decreased diffusion capacity 3 months after the onset of COVID-19 pneumonia

Kyota Shinfuku 1,*, Naoki Takasaka 1, Taiki Fukuda 2, Kentaro Chida 1, Yudai Suzuki 1, Shun Shibata 1, Ayako Kojima 1, Tsukasa Hasegawa 1, Masami Yamada 1, Yumie Yamanaka 1, Yusuke Hosaka 1, Aya Seki 1, Yoshitaka Seki 1, Hiroshi Takeda 1, Takeo Ishikawa 1, Kazuyoshi Kuwano 3
Editor: Samuele Ceruti4
PMCID: PMC9934337  PMID: 36795727

Abstract

Background

Coronavirus disease 2019 (COVID-19) pneumonia can have prolonged sequelae and lead to respiratory dysfunction, mainly because of impaired diffusion capacity for carbon monoxide (DLCO). The clinical factors associated with DLCO impairment, including blood biochemistry test parameters, remain unclear.

Methods

Patients with COVID-19 pneumonia who underwent inpatient treatment between April 2020 and August 2021 were included in this study. A pulmonary function test was performed 3 months after onset, and the sequelae symptoms were investigated. Clinical factors, including blood test parameters and abnormal chest shadows on computed tomography, of COVID-19 pneumonia associated with DLCO impairment were investigated.

Results

In total, 54 recovered patients participated in this study. Twenty-six patients (48%) and 12 patients (22%) had sequelae symptoms 2 and 3 months after, respectively. The main sequelae symptoms at 3 months were dyspnea and general malaise. Pulmonary function tests showed that 13 patients (24%) had both DLCO <80% predicted value (pred) and DLCO/alveolar volume (VA) <80% pred, and appeared to have DLCO impairment not attributable to an abnormal lung volume. Clinical factors associated with impaired DLCO were investigated in multivariable regression analysis. Ferritin level of >686.5 ng/mL (odds ratio: 11.08, 95% confidence interval [CI]: 1.84–66.59; p = 0.009) was most strongly associated with DLCO impairment.

Conclusions

Decreased DLCO was the most common respiratory function impairment, and ferritin level was a significantly associated clinical factor. Serum ferritin level could be used as a predictor of DLCO impairment in cases of COVID-19 pneumonia.

Introduction

The coronavirus disease (COVID-19) pandemic has resulted in more than 614 million confirmed cases with more than 6.5 million deaths as of October 2, 2022 [1]. Owing to the development of vaccines, infections are declining in areas where vaccination coverage is high. However, the uneven distribution of vaccines by region and the emergence of mutant strains of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused the pandemic to persist. SARS-CoV-2 infects the upper respiratory tract, bronchiolar epithelial cells, alveolar epithelial cells, alveolar macrophages, and vascular endothelium via angiotensin-converting enzyme 2 [2]. Excessive immune response and the infecting virus dose are thought to be major factors contributing to the severity of the disease [3]. Dexamethasone, tocilizumab, and baricitinib can be used to suppress the excessive immune response.

In addition, the sequelae after COVID-19 are often prolonged and persist even after viral excretion is stopped. At 60 days after discharge, 87.4% of patients were reported to have symptoms of sequelae, primarily general malaise and dyspnea [4]. In patients who have recovered from COVID-19, reduced diffusion capacity for carbon monoxide (DLCO) has been reported as one of the most frequent respiratory dysfunctions [5, 6]. This abnormality has also been observed in survivors of severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS) [7, 8]. Reduced DLCO has been reported in COVID-19 survivors at the time of discharge and various times thereafter. Mo et al. [9] reported that 47% of COVID-19 survivors showed DLCO impairment at discharge. Previous studies have reported that DLCO impairment remained in 16–82% of cases 3 months after discharge [5, 10, 11]. The severity of COVID-19 and the spread of abnormal chest shadows on computed tomography (CT) scans in COVID-19 pneumonia cases have been reported to be associated with DLCO reduction [12–14]. D-dimer has been reported as a blood biochemical marker [5]. The clinical factors associated with DLCO impairment after COVID-19 pneumonia have not yet been fully clarified. The purpose of this study was to investigate sequelae symptoms and respiratory dysfunction after hospitalization for COVID-19 pneumonia and to identify clinical factors associated with DLCO impairment.

Methods

Study design

This retrospective study was conducted in patients who received inpatient treatment between April 2020 and August 2021 at the Jikei University Daisan Hospital. All COVID-19 diagnoses were based on SARS-CoV2 by real-time reverse transcriptase-polymerase chain reaction, transcription reverse-transcription concerted reaction, or antigen testing. Enrolled patients were those who were hospitalized with COVID-19 pneumonia during the period and who requested outpatient follow-up after the sequelae symptoms and respiratory dysfunction were explained to them. From the standpoint of infection control, an outpatient examination and CT scan were performed 2 months later and it was confirmed that the patient was improving. Then, pulmonary function tests were performed 3 months later. Severity was defined according to the COVID-19 treatment guidelines of the National Institute of Health [15]. In this study, patients were classified into three groups according to the aforementioned guidelines. Groups 1, 2, and 3 included patients who were hospitalized but did not require oxygen supplementation; those who were hospitalized and required conventional oxygen; and those who were hospitalized and required high-flow nasal cannula, noninvasive ventilation, invasive mechanical ventilation, or extracorporeal membrane oxygenation, respectively. Data were collected on patients’ clinical characteristics, blood test results, pulmonary function test, and chest CT images.

Blood test results

Blood tests were performed on admission and periodically thereafter throughout hospitalization. Then, we evaluated the blood sample data during the hospitalization period that may be related to the severity of COVID-19. The peak values of white blood cells, aspartate aminotransferase (AST), alanine aminotransferase (ALT), total-bilirubin, creatine, lactate dehydrogenase (LDH), C-reactive protein, erythrocyte sedimentation rate, ferritin, KL-6, and D-dimer, as well as the minimum values for platelets and hemoglobin, were recorded [16].

Pulmonary function test (PFT)

PFT was performed 3 months after onset by a professional inspection engineer using a FUDAC-7 PFT system (Fukuda Denshi Co. Ltd., Tokyo, Japan). The recorded parameters included the following: total lung capacity (TLC), forced vital capacity (FVC), residual volume, forced expiratory volume in the first second (FEV1), FEV1/FVC ratio, DLCO, and DLCO/alveolar volume (VA). In this study, cases with both DLCO <80% predicted value (pred) and DLCO/VA <80% pred were defined as true DLCO impairment, unaffected by lung volume.

Chest CT

Chest high-resolution CT was performed on all patients using a helical scanner (SOMATOM Definition AS +, Siemens, Erlangen, Germany) with a 1.0-mm slice thickness. Participants underwent chest CT on admission and in the outpatient department 2 months after onset. One radiologist and two pulmonologists, all with more than 10 years of experience, reviewed CT images without knowledge of the patient’s clinical information. For each evaluated CT image, differences in evaluations were settled by consensus. The major CT findings were described using standard international nomenclature defined by the Fleischer Society glossary using terms including ground-glass opacities, consolidation, and reticular pattern [17]. A semiquantitative scoring was based on the chest CT total severity score (TSS) according to the percentage involvement of these abnormalities in each lobe [18, 19]. Each of the five lung lobes was scored on a 5-point scale as follows: 0, indicating no involvement; 1, <25% involvement; 2, 26–49% involvement; 3, 50–75% involvement; and 4, >75% involvement. TSS was the sum of the individual lobar scores and ranged from 0 to 20 points. In addition, the presence or absence of fibrosis on CT image was evaluated on chest CT examination performed at 2 months after onset, based on the presence of traction, parenchymal bands, or honeycombing [20]. Fibrosis was defined as present according to the CT findings when there were two or more of these findings.

Statistical analysis

Data were presented as number (percentage), mean (standard deviation, SD), or median (interquartile range, IQR) depending on whether they were normally or non-normally distributed based on the Shapiro-Wilk test. Fisher’s exact test or the chi-square test was used to compare categorical values between groups, and t-tests or the Mann-Whitney U test and ANOVA or the Kruskal-Wallis test were used to compare continuous variables, depending on whether the data were parametric or non-parametric, respectively. Receiver operating characteristic (ROC) curve analysis and area under the curve (AUC) were used to assess the risk factors associated with DLCO impairment. The cutoff value for each risk factor was determined using Youden’s Index. Multivariable logistic regression models were used to examine the association between clinical factors with cutoff values and DLCO impairment. The statistical analyses were performed using GraphPad Prism version 8.4.3 for Macintosh (GraphPad Software La Jolla, CA, USA) and SPSS version 24 (IBM Corp., Armonk, NY, USA), and the statistical significance was set at p<0.05.

Ethics statement

The study was conducted in accordance with the Declaration of Helsinki. The protocol was approved by the Ethics Committee of Jikei University School of Medicine [No. 30-003(9024)]. In accordance with the ethical guidelines of the Jikei University School of Medicine, informed consent was not necessary for this retrospective study, and opt-out consent was provided on the university website.

Results

Patient characteristics and symptoms of sequelae

From April 2020 to August 2021, 362 patients were admitted to our hospital. Of these, 54 patients who underwent outpatient examinations were included in this study (Table 1). The mean (SD) age was 58.8 (14.8) years, and 35 patients (65%) were male. In comparison between the three groups, age was found to be higher in the oxygen administration groups (p<0.001). All patients were Asian by race. The major complications were hypertension (19 patients, 35%), diabetes mellitus (12 patients, 22%), and respiratory diseases (6 patients, 11%). Respiratory diseases included well-controlled bronchial asthma, pneumothorax, and sleep apnea syndrome, and were not considered to significantly affect PFTs.

Table 1. Characteristics of patients and comparison by severity.

    Group1: Hospitalized but Group2: Hospitalized and Group 3: Hospitalized and  
dose not requires oxygen require conventional oxygen requires HFNC, NIV or IMV,
Parameters Total (n=54) supplementation (n=25) (n=22) ECMO (n=7) p-value
Age (years) a 58.8 ± 14.8 50.5 ± 13.7 67.5 ± 12.5 60.8 ± 8.7 <0.001
Sex, (%Male) b 35 (65) 13 (52) 15 (68) 7 (100) 0.057
Race b
Asian 54 (100) 25 (100) 22 (100) 7 (100) NA
BMI (kg/m2) a 25.2 ± 4.1 26.1± 4.6 24.7 ± 3.9 23.4 ± 2.1 0.259
Smoking history b 25 (46) 11 (44) 11 (50) 3 (43) 0.904
Comorbidities b
Hypertension 19 (35) 2 (8) 14 (64) 3 (43) <0.001
Diabetes melitus 12 (22) 2 (8) 8 (36) 2 (29) 0.059
Respiratory diseases † 6 (11) 4 (16) 1 (5) 1 (14) 0.441
Clinical course c
Hospital period (days) 13.5 (10-17) 11 (9-13.5) 14 (13-17) 24 (17-46) <0.001
Frequency of blood test (days apart) 2.6 (2.2-3) 3 (2.5-3.1) 2.5 (2.3-2.8) 1.7 (1.6-2.2) <0.001
Time from onset to examination a
Chest CT after discharge (days) 51.9 ± 8.6 48.8 ± 7.7 51.7 ± 5.0 63.2 ± 11.9 0.002
Lung function (days) 95.3 ± 15.5 97 ± 18.1 94.4 ± 13.6 92.2 ± 12.3 0.851
Sequelae symptoms b
after 2 months 26 (48) 14 (56) 6 (27) 6 (86) 0.014
dyspnea 13 (24) 7 (28) 3 (14) 3 (42)
cough・sputum 5 (9) 2(8) 1 (5) 2 (29)
general malaise 4 (7) 1 (4) 2 (9) 1(14)
hair loss 2 (4) 2 (8) 0 0
memory loss 1 (2) 1(4) 0 0
chest tightness 1 (2) 1(4) 0 0
after 3 months 12 (22) 6 (24) 2 (9) 4 (57) 0.027
dyspnea 5 (9) 2 (8) 0 3 (43)
cough・sputum 0 0 0 0
general malaise 3 (6) 1 (4) 1(5) 1 (14)
hair loss 2 (4) 2 (8) 0 0
memory loss 1 (2) 0 1(5) 0
chest tightness 1 (2) 1(4) 0 0
Lung function
Lung volume
TLC % pred a 104.4 ± 15.74 109.6 ± 15.85 103.1 ± 11.33 90.07 ± 19.46 0.010
TLC < 80% pred b 4 (7) 1 (4) 0 (0) 3 (43) <0.001
RV % pred a 107 ± 24.94 106.3 ± 28.5 108.9 ± 21.95 103.4 ± 23.05 0.870
Spirometry
FVC % pred a 106.6 ± 16.75 111.8 ± 16.16 104.4 ± 13.92 94.44 ± 21.23 0.035
FVC < 80% pred b 4 (7) 1 (4) 1 (5) 2 (29) 0.072
FEV1.0% pred a 106.1 ± 17.83 101.6 ± 15.2 113.2 ± 17.93 100.1 ± 21.08 0.048
FEV1.0% < 80% pred b 4 (7) 1 (4) 1 (5) 2 (29) 0.072
FEV1.0 / FVC a 79.2 ± 6.59 78.02 ± 7.25 79.32 ± 6.18 83.04 ± 4.03 0.206
FEV1.0/FVC <70% b 5 (9) 2 (8) 3 (14) 0 (0) 0.531
Diffusion capacity
DLCO % pred a 81.56 ± 22.54 80.79 ± 17.01 87.75 ± 25.1 64.86 ± 25.63 0.061
DLCO < 80% pred b 28 (52) 14 (56) 9 (41) 5 (71) 0.316
DLCO/VA % pred a 90.53 ± 16.43 97.96 ± 14.92 85.55 ± 13.26 79.66 ± 20.47 0.004
DLCO/VA < 80% pred b 20 (37) 4 (16) 12 (55) 4 (57) 0.012
DLCO < 80% pred and DLCO/VA < 80% pred b 13 (24) 3 (12) 6 (27) 4 (57) 0.042
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Data are presented as

a mean ± SD

b n (%), or c median (range quartile), NA: not applicable.

†: All four group 1 cases were of bronchial asthma; In group 2, there was one case of pneumothorax, whereas, in group 3, there was one case of sleep apnea syndrome.

HFNC: high-flow nasal cannula, NIV: noninvasive ventilation, IMV: invasive mechanical ventilation, ECMO: extracorporeal membrane oxygenation, BMI: body mass index, TLC: total lung capacity, RV: residual volume, FVC: forced vital capacity, FEV1.0: forced expiratory volume in the first second. DLCO: diffusion capacity for carbon monoxide, VA: alveolar volume.

Two months after onset, 26 patients (48%) were left with sequelae, and 3 months after onset, 12 patients (22%) had residual symptoms. Details of the symptoms are shown in Table 1. The main sequelae symptoms at 3 months were dyspnea and general malaise. The frequency of sequelae at both 2 and 3 months was higher in group 3 than in the other groups (p = 0.014 and p = 0.027, respectively).

Pulmonary function 3 months after COVID-19 onset

Pulmonary function tests 3 months after onset showed a decrease in TLC <80% pred in 4 patients (7%), restrictive impairment in FVC <80% in 4 patients (7%), and obstructive impairment in FEV 1.0/FVC <70% in 5 patients (9%). Twenty-eight patients (52%) had DLCO <80% pred and 20 patients (37%) had DLCO/VA <80% pred. There were 13 patients (24%) who fulfilled both DLCO <80% pred and DLCO/VA <80% pred, and had DLCO impairment unaffected by lung volume abnormality (Table 1).

Comparing pulmonary function according to severity, TLC % pred and FVC % pred were significantly lower in patients with more severe disease. With regard to diffusion impairment, DLCO % pred was lower in group 3, but the difference was not statistically significant. DLCO/VA decreased significantly according to disease severity (p = 0.004), and the proportion of patients who met both DLCO <80% pred and DLCO/VA <80% pred and had DLCO impairment not attributable to an abnormal lung volume was significantly higher in patients with more severe disease (p = 0.042) (Table 1).

Clinical factors related to DLCO impairment

The risk factors associated with DLCO impairment are shown in Table 2. Regarding patient characteristics, the prevalence of smoking history was higher in the DLCO-impaired group (p = 0.023). Regarding clinical course and treatment, the rate of mechanical ventilator use was higher in the DLCO-impaired group (p = 0.049). Regarding the laboratory test results, serum AST (p = 0.034), LDH (p = 0.024), and ferritin (p = 0.001) level were significantly higher in the DLCO-impaired group. With regard to the chest CT findings, both TSS on admission (p = 0.032) and at 2 months (p = 0.005) were higher in the DLCO-impaired group. The prevalence of fibrosis was significantly higher in the DLCO-impaired group (p = 0.005).

Table 2. Characteristics of patients in the DLCO normal group and DLCO-impaired group 3 months after onset.

Parameters DLCO normal group (n=41) DLCO impaired group (n=13) p-value
Age (years) a 59.2 ±15.7 57.2 ±12.1 0.524
Sex, (%Male) b 25 (59) 11 (85) 0.107
BMI (kg/m2) a 25.1± 4.2 25.2 ± 3.9 0.982
Smoking history b 15 (37) 10 (77) 0.023
Time from onset to examination a
Lung function (days) 95.5 ± 17.1 94.7 ± 9.8 0.737
Severity b
Group 1 22 (54) 3 (23) 0.042
Group 2 16 (39) 6 (46)
Group 3 3 (7) 4 (31)
Comorbidity b
Hypertension 15 (37) 4 (31) >0.999
Diabetes melitus 9 (22) 3 (23) >0.999
Respiratory diseases 5 (12) 1 (8) >0.999
Sequelae symptoms b
after 2 months 20 (49) 6 (46) >0.999
after 3 months 8 (20) 4 (31) 0.452
Clinical course and treatment
Hospital period (days) c 13 (10-16) 15 (11-19.5) 0.143
Dexamethasone b 11 (27) 4 (31) >0.999
Remdesivir b 7 (17) 4 (31) 0.429
Heparine b 4 (10) 2 (15) 0.622
Mechanical ventilator use† b 3 (7) 4 (31) 0.049
Laboratory data
WBC (/μL) c 7200 (5300-9000) 8300 (7200-10400) 0.079
Platelets (×104/μL) c 15.2 (12.95-19.05) 16.8 (13.05-20.7) 0.585
Hemoglobin (/μL) c 13.50 (11.8-14.6) 13.1 (11.75-13.85) 0.385
AST (U/L) c 39 (29.5-64.5) 61 (42-124) 0.034
ALT (U/L) c 45 (29-72.5) 98 (40-154) 0.054
T-Bil (mg/dL) c 0.6 (0.5-0.85) 0.75 (0.6-0.97) 0.145
Cr (mg/dL) a 0.93 ± 0.25 1.04 ± 0.26 0.225
LDH (U/L) c 266.5 (225.5-357) 379 (288.5-529) 0.024
CRP (mg/dL) c 4.61 (1.87-10.42) 8.16 (4.89-12.59) 0.151
ESR (mm/hr) a 33.72 ± 20.44 38.64 ± 22.07 0.496
Ferritin (ng/mL) c 407 (178.5-692.5) 1024 (754.5-2258) 0.001
KL-6 (U/mL) c 243 (192-288) 244.5 (165.5-820) 0.674
D-dimer (μg/mL) c 1.1 (0.8-1.7) 1.2 (0.8-3.5) 0.337
Evaluation of chest CT
TSS (admission) c 5 (3-5.5) 6 (4.5-10) 0.032
TSS (2months after onset) c 3 (0-5) 6 (3.5-8) 0.005
Presence of fibrosis (2months after onset) c 9 (21.9) 9 (69.2) 0.005
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Data are presented as a mean ± SD, b n (%), or c median (range quartile).

†: Mechanical ventilator includes high-flow nasal cannula, noninvasive ventilation, invasive mechanical ventilation, and extracorporeal membrane oxygenation.

BMI: body mass index, WBC: white blood cell, AST: aspartate aminotransferase, ALT: alanine aminotransferase, T-Bil: total-bilirubin, Cr: creatinine. LDH: lactate dehydrogenase, CRP: C-reactive protein, ESR: erythrocyte sedimentation rate, CT: computed tomography, TSS: total severity score.

ROC analysis of risk factors associated with DLCO impairment

We then used the ROC curve to examine the AUC and cutoff values by Youden Index for clinical factors that were found to be different in the univariate analysis. Table 3 and Fig 1 show the AUCs and cutoff values for the clinical factors. The AUC for ferritin level was highest: it was 0.783 (95% confidence interval [CI]: 0.616–0.949; p = 0.002). The cutoff value was 686.5 ng/mL with a sensitivity of 84.6% and specificity of 75%. The AUCs for AST and LDH were lower, with cutoff values of 49.5 U/L and 273.5 U/L, respectively. The ROC analysis showed that the cut-offs for TSS at admission and after 2 months were 6.5 and 5.5, respectively, and the AUC was higher for TSS after 2 months, at 0.745 (95% CI: 0.566–0.924; p = 0.008), than that at admission. The AUC for the presence of fibrosis at 2 months was 0.734 (95% CI: 0.569–0.899; p = 0.012).

Table 3. ROC curve and cut-off value for each risk factor.

Risk factors AUC (CI) p-value cut off value sensitivity specificity
AST (U/L) 0.689 (0.519-0.859) 0.042 49.5 0.769 0.650
LDH (U/L) 0.702 (0.537-0.867) 0.030 273.5 0.923 0.500
Ferritin (ng/mL) 0.783 (0.616-0.949) 0.002 686.5 0.846 0.750
TSS (admission) 0.687 (0.499-0.874) 0.045 6.5 0.462 0.875
TSS (2months after onset) 0.745 (0.566-0.924) 0.008 5.5 0.538 0.925
Presence of fibrosis (2months after onset) 0.734 (0.569-0.899) 0.012 ・・・ 0.692 0.780
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ROC: receiver operating characteristic, AUC: area under the curve, CI: confidence interval, AST: aspartate aminotransferase, LDH: lactate dehydrogenase, TSS: total severity score.

Fig 1. Receiver operating characteristic curves of clinical factors affecting DLCO impairment in the univariate analysis.

Fig 1

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Among the comparison factors, ferritin had the highest area under the curve. DLCO: diffusion capacity for carbon monoxide.

Clinical factors associated with DLCO impairment by logistic regression

To examine clinical factors during inpatient care associated with DLCO impairment, multivariable logistic regression analysis adjusted for age, sex, and smoking history was performed for AST, LDH, ferritin, and TSS (on admission) using the cutoff values calculated using ROC curves (Table 4). The results showed that ferritin >686.5 ng/mL (odds ratio: 11.08, 95% CI: 1.84–66.59; p = 0.009) was most strongly associated with DLCO impairment.

Table 4. Logistic regression analysis of risk factors for DLCO impairment.

Risk factors Univariate OR (95% CI) p-value Multivariate OR (95% CI)a p-value
AST (> 49.5 U/L) 6.43 (1.52-27.21) 0.011 ・・・ ・・・
LDH (> 273.5 U/L) 12.6 (1.50-106.02) 0.020 ・・・ ・・・
Ferritin (> 686.5 ng/mL) 17.05 (3.22-90.28) 0.001 11.08 (1.84-66.59) 0.009
TSS at admission (> 6.5) 6.17 (1.47-25.96) 0.013 ・・・ ・・・
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a Multivariate analysis adjusted for age, sex, and smoking history.

OR: odds ratio, CI: confidence interval, AST: aspartate aminotransferase, LDH: lactate dehydrogenase, TSS: total severity score.

Discussion

In this study, among 54 inpatients with COVID-19, 26 (48%) and 12 (22%) had sequelae symptoms after 2 and 3 months, respectively. The most frequent symptoms were dyspnea and general malaise. With regard to respiratory dysfunction, DLCO impairment was the most common. The clinical factor most strongly associated with DLCO impairment was the peak ferritin level. In the multivariable analysis, a peak serum ferritin level >686.5 ng/mL was a risk factor for impaired DLCO.

With regard to sequelae symptoms, the high prevalence of dyspnea and general malaise was similar to that in a previous report [4]. The prevalence of residual symptoms was higher in the severe group, which was also similar to that in a previous report [21]. In the present study, only 22% of patients had residual symptoms at 3 months, while 87% of patients in an Italian study had residual symptoms at 2 months and 76% of patients in a Chinese study had residual symptoms at 6 months [4, 21]. Factors contributing to a lower prevalence of sequelae in the present study include the fact that these studies used questionnaires, while the present study was based on interviews during outpatient consultations, and minor or infrequent symptoms may not have been recorded. Then, in our study, all patients were Asian, whereas the Italian study did not provide details but may have included more whites. Racial differences could also contribute to the differences in the proportion of residual symptoms between studies. In addition, the Italian study was from the first wave of the infection, when the efficacy of dexamethasone and antiviral drugs had not been established. Differences in treatment may also have affected residual symptoms.

On pulmonary function tests 3 months after hospital discharge, decreased DLCO was observed in 52% of cases, the most common functional impairment. This finding is consistent with those of previous reports. The percentage of DLCO <80% pred 3 months after hospital discharge has been reported to range from 16% to 82%, depending on disease severity [5, 10, 11]. Furthermore, in this study, the proportion of DLCO <80% pred and DLCO/VA <80% pred was 24%, which showed a stronger correlation with severity than DLCO <80% pred alone, and was thought to be a better indicator of true residual diffusion impairment. In autopsy studies of patients who have died of COVID-19, lung pathology showed diffuse alveolar damage (DAD) findings [22, 23], suggesting that damage to alveolar epithelial cells, basement membrane, and pulmonary capillary endothelium, as well as the associated growth of collagen fibers, occurred in these cases with residual DLCO damage. On the other hand, TLC and FVC decreased with increasing severity, although less frequently (7%) than in DLCO reduction. The finding that TLC decreased in a small number of severe cases is consistent with those of previous studies [9, 10], suggesting that the destruction of alveolar structures by fibrosis associated with DAD and residual atelectasis may be responsible for the decrease.

In a multivariable analysis of clinical factors, peak ferritin level was the factor most strongly associated with DLCO impairment. Apart from COVID-19, there were no cases in this study with a history of autoimmune disease or blood transfusions that contributed to elevated ferritin levels. Previous reports on blood biochemistry have shown an association with the D-dimer level [5]. No other blood biochemical markers associated with respiratory dysfunction have been reported. Serum ferritin level has already been shown to be associated with COVID-19 severity and mortality [24]. However, to the best of our knowledge, no studies have shown a direct association between elevated ferritin level and respiratory dysfunction. This study is the first to suggest a direct association of ferritin level and DLCO impairment with COVID-19 pneumonia. Ferritin consists of a shell protein with iron molecules encapsulated in the center [25]. Ferritin synthesis is enhanced by the production of cytokines [26]. During cytokine storms, the levels of cytokines, such as interleukin (IL)-1β, IL-6, IL-12, interferon, and tumor necrosis factor-α, are elevated and ferritin is secreted from macrophages, the liver, and Kupffer cells. The uncontrolled virus invades the tissues and infects cells in the alveolar epithelium, airway epithelium, and pulmonary capillary endothelium, causing inflammatory cell death and leading to respiratory dysfunction. To cope with the dead inflammatory cells, an even larger number of immune cells infiltrate into the lungs, presumably resulting in excessive production of inflammatory cytokines [27]. Ferritin is presumed to indirectly reflect this cytokine storm. The association between DLCO impairment and ferritin level suggests that elevated ferritin may indicate inflammatory cell death associated with respiratory dysfunction. In this study, a peak serum ferritin level of >686.5 ng/mL was a strong risk factor for DLCO impairment, and careful follow-up examination and rehabilitation intervention may be effective in preventing residual respiratory disability in patients with this finding [28]. It is also possible that elevated ferritin level could be used as a predictor of respiratory dysfunction due to viral pneumonia.

There are limitations to this study. First, it was a retrospective study with a limited number of cases at a single institution. The applicants were included and selection bias is taken into account. A larger prospective study of clinical factors predicting respiratory dysfunction after COVID-19 is needed. Second, there were no pulmonary function tests conducted before the onset of COVID-19. Cases of respiratory diseases associated with DLCO impairment, such as chronic obstructive pulmonary disease and interstitial pneumonia, were not included. Nevertheless, it could not be ascertained whether there was already reduced DLCO before the onset of COVID-19.

Conclusions

DLCO impairment was the most frequent disorder of respiratory function, and the peak serum ferritin level was associated with DLCO impairment. A ferritin level of >686.5 ng/mL could be a risk factor for DLCO impairment and indirectly reflects tissue damage with cytokine storms.

Acknowledgments

We thank Dr. Akiyoshi Kinoshita, Department of Gastroenterology at Jikei University Daisan Hospital, for his help with the statistical analysis. We would also thank all the medical staff, including nurses and laboratory technicians, who are involved in the treatment of COVID-19 patients at our hospital.

Data Availability

All relevant data are within the paper.

Funding Statement

The author(s) received no specific funding for this work.

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Decision Letter 0

Steve Zimmerman

24 Aug 2022

PONE-D-22-09029Association between serum ferritin levels and decreased diffusion capacity 3 months after the onset of COVID-19 pneumoniaPLOS ONE

Dear Dr. shinfuku,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

The manuscript has been evaluated by two reviewers, and their comments are available below.

The reviewers have raised a number of major concerns, including the small sample size, the lack of baseline data, and the possibility of other causes of elevated ferritin levels. Could you please carefully revise the manuscript to address all comments raised?

Please submit your revised manuscript by Oct 08 2022 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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We look forward to receiving your revised manuscript.

Kind regards,

Steve Zimmerman, PhD

Associate Editor, PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Yes

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Small sample size.

Given the absence of baseline lung function measurement, it may be that 13/54 patients already had abnormal DLCO/ DLCO-Va prior to COVID-19.

Abstract-background should define what’s known in the field or invoke interest to your question rather than describe your methods.

With a sample size of 13, it is difficult to make a conclusion that ferritin can be a long term lung function abnormalities in COVID-19. At best, you can report an association.

The manuscript presents no novel findings. How does this paper add to the scientific literature?

Please better clarify CT shadows in introduction. What is CT shadows?

Standard definition of severe and critical COVID-19 has not been used.

How did you decide the blood test results selected? Was it just the data that was available?

If you used CT findings to define your outcomes, mention this in the abstract.

Alternative causes of DLCO reduction were not ruled out.

Present findings in table 2. Table 1 should be demographics.

Please correct typing errors.

Reviewer #2: Though this is a small study, it highlights an important association between ferritin and diffusion capacity in COVID19. It serves to identify patients who may be at a higher risk for developing future interstitial lung disease and also aids in those who may benefit from post COVID PFT surveillance.

Some questions from my review as below:

1) Could not understand how AST, LDH and TSS that were significant in univariate analysis, did not show significance in multivariate analysis? Can explain the reason for this as Table 3 and Table 4 seem to show significant p-values for them for AUC and OR respectively. Is this related to the number of patients?

2) The relationship between DLCO and Ferritin is well documented in conditions such as Thalassemia especially when transfusion is needed. Were there any patients who were transfused? Also were there any underlying inflammatory diseases in the patients such as arthritis, autoimmune conditions that could have elevated the ferritin level?

**********

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Reviewer #1: No

Reviewer #2: Yes: Bright Thilagar

**********

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PLoS One. 2023 Feb 16;18(2):e0281249. doi: 10.1371/journal.pone.0281249.r002

Author response to Decision Letter 0

15 Oct 2022

Reviewer 1

The authors would like to thank the reviewer for their constructive critique to improve the manuscript. We have made every effort to address the issues raised and to respond to all comments. The revisions are indicated in blue font in the revised manuscript. Please, find next a detailed, point-by-point response to the reviewer's comments. We hope that our revisions will meet the reviewer’s expectations.

・Small sample size.

We appreciate the reviewer’s comment on this point. We agree with the reviewer that the sample size was small. However, we believe that it is important to note that the frequency of DLCO impairment was quite high. Based on the reviewer’s comments, we have discussed this issue as a limitation as follows:

“There are some limitations in this study. First, it was a retrospective study with a limited number of cases at a single institution.” (Lines 294–295)

・Given the absence of baseline lung function measurement, it may be that 13/54 patients already had abnormal DLCO/ DLCO-Va prior to COVID-19.

We appreciate the reviewer’s comment on this point. Based on the reviewer’s comments, we have discussed this issue as a limitation as follows:

“Second, there were no pulmonary function tests conducted before the onset of COVID-19. Cases of respiratory diseases associated with DLCO impairment, such as chronic obstructive pulmonary disease and interstitial pneumonia, were not included. Nevertheless, it could not be ascertained whether there was already reduced DLCO before the onset of COVID-19.” (Lines 295–299)

・Abstract-background should define what’s known in the field or invoke interest to your question rather than describe your methods.

We appreciate the reviewer’s comment on this point. Per the reviewer’s insightful suggestion, we have added more background information in the Abstract section as follows:

“The clinical factors associated with DLCO impairment, including blood biochemistry test parameters, remain unclear.” (Lines 23–24)

・With a sample size of 13, it is difficult to make a conclusion that ferritin can be a long term lung function abnormalities in COVID-19. At best, you can report an association.

We appreciate the reviewer’s comment on this point. Based on the reviewer’s suggestion, we have revised the conclusion in the Abstract and in the main text as follows:

“Decreased DLCO was the most common respiratory function impairment, and ferritin level was a significantly associated clinical factor. Serum ferritin level could be used as a predictor of DLCO impairment in cases of COVID-19 pneumonia.” (Lines 39–41)

“DLCO impairment was the most frequent disorder of respiratory function, and the peak serum ferritin level was associated with DLCO impairment. Ferritin level of >686.5 ng/mL could be a risk factor for DLCO impairment and indirectly reflects tissue damage with cytokine storms.” (Lines 302–305)

・The manuscript presents no novel findings. How does this paper add to the scientific literature?

We appreciate the reviewer’s comment on this point. It is known that DLCO impairment is common in COVID-19 pneumonia and that ferritin is associated with severity and mortality. However, to our knowledge, this is the first study to show a direct association between the peak ferritin level and DLCO impairment. We believe that this finding is novel and would contribute to literature. We have highlighted the novelty of the study as follows:

“To the best of our knowledge, no other blood biochemical markers associated with respiratory dysfunction have been reported to date. Moreover, the serum ferritin level has already been shown to be associated with COVID-19 severity and mortality [24]. However, to the best of our knowledge, no studies to date have shown a direct association between the elevated ferritin level and respiratory dysfunction. This study is the first to suggest a direct association of ferritin level and DLCO impairment with COVID-19 pneumonia.” (Lines 277–283)

“It is also possible that elevated ferritin levels could be used as a predictor of respiratory dysfunction due to viral pneumonia.” (Lines 291–293)

・Please better clarify CT shadows in introduction. What is CT shadows?

We appreciate the reviewer’s comment on this point. Based on the reviewer’s comments, we have provided a description in the Introduction section as follows:

“Severity of COVID-19 and the spread of abnormal chest shadows on computed tomography (CT) scans in COVID-19 pneumonia cases have been reported to be associated with DLCO reduction [12-14].” (Lines 67–69)

・Standard definition of severe and critical COVID-19 has not been used.

We appreciate the reviewer’s comment on this point. As the reviewer pointed out, we have revised the severity classification according to the NIH criteria. Based on the reviewer’s comments, we have corrected the description of the study design as follows:

“The severity was defined according to the COVID-19 treatment guidelines of the National Institute of Health [15]. In this study, patients were classified into three groups according to the aforementioned guidelines. Groups 1, 2, and 3 included patients who were hospitalized but did not require oxygen supplementation; those who were hospitalized and required conventional oxygen; and those who were hospitalized and required high-flow nasal cannula, noninvasive ventilation, invasive mechanical ventilation, or extracorporeal membrane oxygenation, respectively.” (Lines 80–87)

Moreover, we have corrected the description in Table 1.

・How did you decide the blood test results selected? Was it just the data that was available?

We would like to thank the reviewer for the questions. To respond to the reviewer’s question, we have added the following part to the revised manuscript:

“Blood tests were performed on admission and periodically thereafter throughout hospitalization. Then, we evaluated the blood sample data during the hospitalization period that may be related to the severity of COVID-19.” (Lines 92–94)

Moreover, we have provided more information concerning the frequency of blood test (days apart) in Table 1.

・If you used CT findings to define your outcomes, mention this in the abstract.

Per the reviewer’s insightful suggestion, we have added the following part to the Abstract:

“Clinical factors, including blood test parameters and abnormal chest shadows on computed tomography, of COVID-19 pneumonia associated with DLCO impairment were investigated.” (Lines 27–29)

・Alternative causes of DLCO reduction were not ruled out.

According to the reviewer’s insightful suggestion, we have added the following part to the Discussion section:

“Second, there were no pulmonary function tests conducted before the onset of COVID-19. Cases of respiratory diseases associated with DLCO impairment, such as chronic obstructive pulmonary disease and interstitial pneumonia, were not included. Nevertheless, it could not be ascertained whether there was already reduced DLCO before the onset of COVID-19.” (Lines 295–299)

・Present findings in table 2. Table 1 should be demographics.

We appreciate the reviewer’s comment on this point. Please note that we have made corrections in Tables 1 and 2. The revised parts are presented in blue font.

・Please correct typing errors.

We would like to apologize to the reviewer for the typing errors. Please note that we have double-checked and corrected the typing in the text and tables.

Reviewer 2

The authors would like to thank the reviewer for their constructive critique to improve the manuscript. We have made every effort to address the issues raised and to respond to all comments. The revisions are indicated in red font in the revised manuscript. Please, find next a detailed, point-by-point response to the reviewer's comments. We hope that our revisions will meet the reviewer’s expectations.

1) Could not understand how AST, LDH and TSS that were significant in univariate analysis, did not show significance in multivariate analysis? Can explain the reason for this as Table 3 and Table 4 seem to show significant p-values for them for AUC and OR respectively. Is this related to the number of patients?

We appreciate the reviewer’s comment on this point. As the reviewer indicated, the small sample size may have had an impact. Moreover, in the present study, multivariate analysis was performed for significant items in univariate terms, with cutoff values set using the ROC curves. The multivariate analysis used a stepwise approach, but only ferritin, which is most associated with DLCO impairment, remained as a variable. AST, LDH, and TSS, which were significant in the ROC curve AUC and univariate analyses, were excluded and did not remain. We thought that AST and LDH would not be significant in the multivariate analysis because of confounding factors, such as liver injury and muscle destruction. We also considered that TSS was removed and did not remain as a final factor because of the stronger association of ferritin with DLCO impairment.

2) The relationship between DLCO and Ferritin is well documented in conditions such as Thalassemia especially when transfusion is needed. Were there any patients who were transfused? Also were there any underlying inflammatory diseases in the patients such as arthritis, autoimmune conditions that could have elevated the ferritin level?

We would like to thank the reviewer for the comment. We again checked the transfusion history of the patients and comorbidities that may have contributed to the elevated ferritin level, such as autoimmune diseases, but none of the cases were applicable. Please note that we have discussed this issue in the revised manuscript as follows: “Apart from COVID-19, there were no cases in this study with a history of autoimmune disease or blood transfusions that contributed to elevated ferritin levels.” (Lines 274–276)

Attachment

Submitted filename: Response_to_Reviewers.doc

Click here for additional data file. (55KB, doc)

Decision Letter 1

Samuele Ceruti

22 Nov 2022

PONE-D-22-09029R1Association between serum ferritin levels and decreased diffusion capacity 3 months after the onset of COVID-19 pneumoniaPLOS ONE

Dear Dr. shinfuku,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

==============================

ACADEMIC EDITOR:

Dear Authors

the paper is extremely interesting and has generated an interesting debate among the reviewers. There are differing opinions on the subject; in this context of debate and extremely conflicting opinions between reviewers, I personally consider the paper worthy of note, although I think it needs to be further refined and clarified.

In particular, taking into account the suggestions of reviewer #3, I would ask you to improve the methodological precision described in the M&M session, particularly in the first part where you describe the groups, explaining well the whole methodology of the construct of your paper. 

Again, personally I have two more considerations. First, as you also suggested in the Discussion, it is possible that the differences you found with respect to the Italian literature are also related to racial differences; however, nowhere do I find an indication of the percentage of the Asian, Caucasian, black population that you analysed. This data needs to be implemented. Secondly, it is also possible that the difference between the Italian study and yours is linked to a different pharmacological treatment (steroids, anticoagulants, anti-IL6, etc.) which had not yet been implemented at the time of the first wave. This consideration must also be implemented too.

I therefore ask you again for more clarity in the methodology (especially in the first part of the M&M session) and the resolution of these two points suggested before.

Please submit your revised manuscript by Jan 06 2023 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

  • A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

  • A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

  • An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Samuele Ceruti

Academic Editor

PLOS ONE

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #2: All comments have been addressed

Reviewer #3: (No Response)

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2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #2: Yes

Reviewer #3: No

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #2: Yes

Reviewer #3: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #2: Yes

Reviewer #3: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #2: Yes

Reviewer #3: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #2: Suggestions were accepted and corrections made. Authors were responsive to the corrections in a reasonable and responsive manner.

Reviewer #3: I was asked to review this revised manuscript in its current form, having not reviewed the initial version.

Overall, I agree with prior reviews in general. This retrospective review of a single institution’s experience reveals that peak serum ferritin measures at the time of admission are the most predictive of a reduced DLCO and DLCO/VA at 3 months. I need to reiterate that the number of subjects is small, and the general lack of clinical characterization of the cohort make the results less than convincing. And there is no description of how these subjects were enrolled. The authors state that the CT was done 2 months after onset, and PFTs were done 3 months after onset. Why? And how can you be so sure that the CTs were done exactly 2 months after and the PFTs done exactly 3 months after? There is no mention of a protocol. There is no discussion of why ferritin levels would correlate with a reduction in DLCO. What mechanism of action would that be? The authors are encouraged to develop a prediction model, using multiple clinical, lab, and radiologic characteristics that could be validated prospectively. But as it stands now, I’m not convinced this adds much to the existing literature regarding reduced DLCO after Covid.

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Reviewer #2: Yes: Bright Thilagar

Reviewer #3: No

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PLoS One. 2023 Feb 16;18(2):e0281249. doi: 10.1371/journal.pone.0281249.r004

Author response to Decision Letter 1

9 Dec 2022

Academic editor

The authors would like to thank the editor for their constructive critique to improve the manuscript. We have made every effort to address the issues raised and to respond to all comments. The revisions are indicated in blue font in the revised manuscript. Revisions that are common to the corrections to the reviewer's remarks are noted in green font. Please, find next a detailed, point-by-point response to the editor's comments. We hope that our revisions will meet the reviewer’s expectations.

・In particular, taking into account the suggestions of reviewer #3, I would ask you to improve the methodological precision described in the M&M session, particularly in the first part where you describe the groups, explaining well the whole methodology of the construct of your paper.

We appreciate the reviewer’s comment on this point. We added a description of the research methods in the study design section as follows: “Enrolled patients were those who were hospitalized with COVID-19 pneumonia during the period and who requested outpatient follow-up after the sequelae symptoms and respiratory dysfunction were explained to them. From the standpoint of infection control, an outpatient examination and CT scan were performed 2 months later and it was confirmed that the patient was improving. Then, pulmonary function tests were performed 3 months later.” (Lines 81-86)

We then added the time to the examination for each group in Tables 1 and 2.

Again, personally I have two more considerations. First, as you also suggested in the Discussion, it is possible that the differences you found with respect to the Italian literature are also related to racial differences; however, nowhere do I find an indication of the percentage of the Asian, Caucasian, black population that you analysed. This data needs to be implemented.

We appreciate the reviewer’s comment on this point. We have added a description of the race of the patients in Table 1. In addition, based on the editor’s comments, we have corrected the description of the discussion section as follows: “Then, in our study, all patients were Asian, whereas the Italian study did not provide details but may have included more whites. Racial differences could also contribute to the differences in the proportion of residual symptoms between studies.” (Line 264-267)

Secondly, it is also possible that the difference between the Italian study and yours is linked to a different pharmacological treatment (steroids, anticoagulants, anti-IL6, etc.) which had not yet been implemented at the time of the first wave. This consideration must also be implemented too.

We appreciate the reviewer’s comment on this point. We have added a description in the discussion section as follows: “In addition, the Italian study was from the first wave of the infection, when the efficacy of dexamethasone and antiviral drugs had not been established. Differences in treatment may also have affected residual symptoms.” (Lines 267-269)

Reviewer 3

The authors would like to thank the reviewer for their constructive critique to improve the manuscript. We have made every effort to address the issues raised and to respond to all comments. The revisions are indicated in red font in the revised manuscript. Revisions that are common to the corrections to the editor's remarks are noted in green font. Please, find next a detailed, point-by-point response to the reviewer's comments. We hope that our revisions will meet the reviewer’s expectations.

Overall, I agree with prior reviews in general. This retrospective review of a single institution’s experience reveals that peak serum ferritin measures at the time of admission are the most predictive of a reduced DLCO and DLCO/VA at 3 months. I need to reiterate that the number of subjects is small, and the general lack of clinical characterization of the cohort make the results less than convincing.

And there is no description of how these subjects were enrolled. The authors state that the CT was done 2 months after onset, and PFTs were done 3 months after onset. Why? And how can you be so sure that the CTs were done exactly 2 months after and the PFTs done exactly 3 months after? There is no mention of a protocol.

We appreciate the reviewer’s comment on this point. We added a description of how the eligible patients were enrolled and how the CT scan was performed after 2 months and the pulmonary function test after 3 months in the study design section as follows: “Enrolled patients were those who were hospitalized with COVID-19 pneumonia during the period and who requested outpatient follow-up after the sequelae symptoms and respiratory dysfunction were explained to them. From the standpoint of infection control, an outpatient examination and CT scan were performed 2 months later and it was confirmed that the patient was improving. Then, pulmonary function tests were performed 3 months later.” (Lines 81-86)

We then added the time to the examination for each group in Tables 1 and 2.

There is no discussion of why ferritin levels would correlate with a reduction in DLCO. What mechanism of action would that be?

We appreciate the reviewer’s comment on this point. We added a description of the association between ferritin levels and DLCO reduction in the discussion section as follows:

“The uncontrolled virus invades the tissues and infects cells in the alveolar epithelium, airway epithelium, and pulmonary capillary endothelium, causing inflammatory cell death and leading to respiratory dysfunction. To cope with the dead inflammatory cells, an even larger number of immune cells infiltrate into the lungs, presumably resulting in excessive production of inflammatory cytokines [27]. Ferritin is presumed to indirectly reflect this cytokine storm. The association between DLCO impairment and ferritin level suggests that elevated ferritin may indicate inflammatory cell death associated with respiratory dysfunction.” (Lines 299-306)

The authors are encouraged to develop a prediction model, using multiple clinical, lab, and radiologic characteristics that could be validated prospectively. But as it stands now, I’m not convinced this adds much to the existing literature regarding reduced DLCO after Covid.

We appreciate the reviewer’s comment on this point. We added a corresponding description in the discussion section as follows: “First, it was a retrospective study with a limited number of cases at a single institution. The applicants were included and selection bias is taken into account. A larger prospective study of clinical factors predicting respiratory dysfunction after COVID-19 is needed.” (Line 311-314)

Attachment

Submitted filename: Response_to_Reviewers.doc

Click here for additional data file. (44.5KB, doc)

Decision Letter 2

Samuele Ceruti

19 Jan 2023

Association between serum ferritin level and decreased diffusion capacity 3 months after the onset of COVID-19 pneumonia

PONE-D-22-09029R2

Dear Dr. shinfuku,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Kind regards,

Samuele Ceruti

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Dear Authors,

I'm so sorry about this delay in my answer, but as you can read below, there was a lot of debate at the reviewers' level; although one of the reviewers requested that the paper be rejected, I feel that the reasons given are not sufficient to reject the paper, especially, after the authors have taken care of the initial comments.

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #2: All comments have been addressed

Reviewer #3: (No Response)

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2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #2: Yes

Reviewer #3: Partly

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3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #2: Yes

Reviewer #3: Yes

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4. Have the authors made all data underlying the findings in their manuscript fully available?

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Reviewer #2: Yes

Reviewer #3: Yes

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Reviewer #2: Yes

Reviewer #3: Yes

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6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #2: Although not perfect and from a single institution, I think this data would add to the existing literature on supporting follow up DLCO in post COVID patients and is important in Long COVID research.

Reviewer #3: Thank you for revising the manuscript according to my comments. Upon re-review of the manuscript, you have addressed mostly the concerns that I had upon first review. However, your finding that low DLCO is the most common PFT abnormality is not new, and this small study does not add anything to the established literature on the topic.

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If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #2: Yes: Bright Thilagar

Reviewer #3: No

**********

Acceptance letter

Samuele Ceruti

5 Feb 2023

PONE-D-22-09029R2

Association between serum ferritin level and decreased diffusion capacity 3 months after the onset of COVID-19 pneumonia

Dear Dr. Shinfuku:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Samuele Ceruti

Academic Editor

PLOS ONE

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    Submitted filename: Response_to_Reviewers.doc

    Click here for additional data file. (55KB, doc)
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    Submitted filename: Response_to_Reviewers.doc

    Click here for additional data file. (44.5KB, doc)

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    All relevant data are within the paper.

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