Detection of Serum KL-6 and SARS-CoV-2 Antibody in Patients with Coronavirus Disease 2019 and the Diagnostic Value in Severe Disease

Background: Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a signicant threat to human health, but its clinical manifestations vary greatly among individuals. Early detection and treatment are important for severely ill patients to improve their prognosis and reduce the risk of death. Methods: In the present study, serum markers were detected and analyzed in moderately ill and severely ill patients. Results: The results found that there were statistically signicant differences in age, serum Krebs von den Lungen-6 (KL-6) and Immunoglobulin A (IgA) levels between severely ill patients and moderately ill patients (P < 0.05). The cut-off of using KL-6 alone for the diagnosis of severely ill patients was 298.91 U/mL, with an AUC of 0.737, a sensitivity of 100%, and a specicity of 43%. When the diagnosis was performed using KL-6 in combination with Interleukin-6 (IL-6), an indicator of infection, the AUC was 0.776, with a sensitivity and specicity of 82% and 69%, respectively. When the three above were used in combination for diagnosis, the AUC was 0.785, and the sensitivity and specicity were 100% and 59%, respectively. After rehabilitation, the serum levels of KL-6, C-reactive protein (CRP), as well as antibodies, IgA, IgM and IgG, were signicantly lower than those in the early stage of hospitalization. Conclusion: In the present study, KL-6 and IgA were found to have some diagnostic ecacy for severely ill patients with COVID-19, but larger cohort studies are still needed for further conrmation, which in turn improves the diagnostic and therapeutic eciency of severely ill patients. GM-CSF: Granulocyte-macrophage factor.


Background
Coronavirus disease 2019 (COVID-19) is a global infectious disease caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which poses a threat to human health [1,2]. The clinical manifestations of patients with coronavirus disease 2019 vary greatly, from no symptoms in patients with asymptomatic infection to dry cough, fever, and nasal congestion in moderately ill patients, while severely ill patients may present with chest tightness, asthma, and dyspnea, and critically ill patients may have secondary multiple organ dysfunction and the disease can be fatal [3][4][5][6]. Most moderately ill patients can recover after symptomatic and supportive treatment, while COVID-19 in severely ill/critically ill patients can be life-threatening due to a series of rapid pathophysiological changes such as body in ammatory response and "cytokine storm" [7][8][9]. Current diagnostic and therapeutic regimens emphasize early evaluation, and early treatment of severely ill/critically ill patients to help improve patient prognosis and reduce the risk of death [10][11][12][13]. We previously reported serum SARS-CoV-2 speci c Immunoglobulin A (IgA) is positively correlated with disease severity [14], and Krebs von den Lungen-6 (KL-6) is a good indicator for lung injury and in ammation in COVID-19 patients [15].
In the present study, we detected and analyzed the serum markers of moderately ill and severely ill patients to explore the value of each indicator in assessing the severity of patients, assisting in the diagnosis of severely ill patients, and improving the e ciency of diagnosis and treatment.

Statistical methods
Statistical analysis of data was performed using the SPSS 22.0 software package. Data are presented as counts and percentages for categorical data and medians and interquartile ranges (IQRs) for continuous data. χ 2 -test was used for intergroup comparison of categorical data, t-test was used for intergroup comparison of measurement data conforming to normal distribution, and Mann-Whitney U-test was used for measurement data not conforming to normal distribution. The diagnostic e cacy of serum marker levels in severely ill patients was analyzed using receiver operating characteristic (ROC) curves, and the area under the ROC curve (AUC) was used to evaluate the diagnostic e cacy. The sensitivity and speci city were selected according to the optimal screening cut-off value when the Youden index was the largest. P < 0.05 was considered statistically signi cant.

Patients' basic characteristics
As shown in Table 1, there were 52 moderately ill patients (26 males and 26 females) and 12 severely ill patients (9 males and 3 females) among 64 patients with con rmed diagnosis. There was no signi cant difference in gender across different types of COVID-19 (χ 2 = 2.459, P = 0.117). The mean age of moderately ill patients was 42 (31, 51.75) years, and the age of severely ill patients was 53.5 (49, 66.75) years. The age was signi cantly different across different types of COVID-19 (t = − 3.786, P < 0.001). Comparison of serum marker detection results between severely ill and moderately ill patients The results of serum biomarkers in the early stage of hospitalization are shown in Table 2. The levels of KL-6 and IgA were signi cantly elevated in severely ill patients compared with moderately ill patients, and the difference was statistically signi cant (P < 0.05). In addition, the levels of serum IL-6, IgM and IgG were also higher in severely ill patients than in moderately ill patients, but the difference was not statistically signi cant (P > 0.05). Analysis of the diagnostic e cacy of serum markers in severely ill patients ROC curves were used to analyze the diagnostic e cacy of using various parameters for diagnosing severely ill patients with COVID-19, and the single test of KL-6 had the best e cacy in diagnosing severely ill patients, followed by IL-6 and IgA (Fig. 1). The cut-off of using KL-6 alone for the diagnosis was 298.91 U/mL, with an AUC of 0.737, a sensitivity of 100%, and a speci city of 43%. When the two tests were used in combination for diagnosis, KL-6 combined with IL-6 showed the best diagnostic e cacy, with an AUC of 0.776 and a sensitivity and speci city of 82% and 69%, respectively. The AUC of the combined diagnosis of the three tests of KL-6, IL-6 and IgA was 0.785, with a sensitivity and speci city of 100% and 59%, respectively.

Results of serum marker tests in patients at different stages
The patients had changes in the levels of multiple serum biomarkers at 3-month follow-up compared with early hospitalization, as shown in Table 3. Among them, CRP, an in ammatory indicator, and KL-6, an indicator of pulmonary brosis, were signi cantly reduced, suggesting that the patients' condition had improved. Notably, the levels of all three antibodies, IgA, IgM, and IgG, were signi cantly reduced, and the differences were statistically signi cant (P < 0.001).

Discussion
The clinical features of COVID-19 patients are diverse, with some patients having mild symptoms and some progressing into severe cases, which can be life-threatening. Early identi cation of severely ill patients will be bene cial to improve patient's prognosis and reduce mortality. In the present study, we investigated potential biomarkers in severely ill patients by analyzing the results of serological index indicators in patients at the early stage of hospitalization.
The results of the present study showed that serum KL-6 and IgA levels were signi cantly elevated in severely ill patients compared with moderately ill patients. KL-6 is a high-molecular-weight mucin expressed by type II alveolar epithelial cells (AECII). When AECII is injured, more KL-6 is secreted through its proliferation and differentiation and leaks through the basement membrane into the blood circulation, thereby increasing KL-6 levels in serum [16,17]. KL-6 is one of the important markers of pulmonary brosis, and KL-6 is elevated in the serum of patients with interstitial pneumonia, and the degree of elevation has a correlation with the occurrence and severity of interstitial lung disease [18][19]. d'Alessandro et al. also con rmed that the serum KL-6 could diagnose severe COVID-19, with a cut-off of 406.5 U/ml, an AUC of 0.824, and a sensitivity and speci city of 83% and 89%, respectively [20]. In the report of Awano et al., serum KL-6 value of 371 U/ml was used as the optimal cut-off to evaluate disease severity with a sensitivity of 85.7% and speci city of 96.6% [21]. In the present study, the cut-off of using KL-6 alone for the diagnosis of severely ill patients was 298.91 U/mL, with an AUC of 0.737, a sensitivity of 100%, and a speci city of 43%. Although the sensitivity was good, the speci city was low, and the cutoff was also different from the ndings of d'Alessandro et al [20]. The diagnostic e cacy of KL-6 for severe disease in patients with COVID-19 remains to be con rmed by larger cohort studies.
IL-6 is a commonly used rapid detection indicator of infectious diseases and one of the important in ammatory factors triggering cytokine storm in patients with COVID-19 [22,23]. After SARS-CoV-2 virus infection, pathogenic T cells are rapidly activated and factors such as Granulocyte-macrophage Colony Stimulating Factor (GM-CSF) and IL-6 are abundantly produced, thus forming an in ammatory storm and leading to severe immune damage in lungs and other organs [24]. IL-6 levels progressively increase in severely ill patients with COVID-19 and have been demonstrated to be an early warning indicator of progression into severe disease [25]. In the present study, the serum IL-6 levels were elevated in severely ill patients compared with moderately ill patients, but the difference was not statistically signi cant (Table 2). However, the results of ROC curve analysis still showed that IL-6 had some e cacy in diagnosing severely ill patients: The AUC was 0.720, and the sensitivity and speci city were 64% and 80%, respectively. IL-6 had better diagnostic e cacy combined with KL-6, with an AUC of 0.776 and sensitivity and speci city of 82% and 69%, respectively. The present study also found that IgA was signi cantly elevated in severely ill patients with COVID-19 and could be used as one of the potential markers for the diagnosis of progression into critical disease, and the results were consistent with the results of the study conducted by Ma et al. [14].
At the three-month follow-up, a number of serum marker levels changed in patients with coronavirus disease 2019, suggesting that the patients' condition had recovered. In addition to a signi cant decrease in the level of the in ammatory factor CRP, KL-6, an indicator of pulmonary brosis, was also signi cantly reduced. In addition, all three antibodies speci c to SARS-CoV-2, IgA, IgM, and IgG, were very signi cantly down-regulated, consistent with the results reported in previous studies [14,26]. It is worth investigating whether recovered patients will have the same risk of infection as that of previously uninfected people when encountering the SARS-CoV-2 virus again [26].
In summary, close monitoring and tracking of serum KL-6, IL-6 and IgA, three potential risk indicators of severe disease, and early intervention will likely avoid the progression into severe disease of COVID-19 and improve patient prognosis, which has important clinical signi cance. However, disease progression is an ongoing process, and the present study is limited by no continuous monitoring in patients due to condition restrictions. In addition, although the present study suggested that KL-6, IL-6 and IgA had potential value in the diagnosis of progression into severe coronavirus disease 2019, the limitations of this study, which include the limited number of cases and the retrospective nature of study, indicate that this value needs to be con rmed by a larger cohort study.

Conclusions
KL-6 and IgA were found to have some diagnostic e cacy for severely ill patients with COVID-19 in this study, but larger cohort studies are still needed for further con rmation, which in turn improves the diagnostic and therapeutic e ciency of severely ill patients.