The Relation between Immunological Features and the Positive SARS-CoV-2 Nucleic Acid in Patients with Nonsevere COVID⁃19

Objective The novel coronavirus nucleic acid results are the core indicators of illness monitoring. This study aimed to evaluate the relationship between immunological features and positive SARS-CoV-2 nucleic acid by analyzing the clinical and immunological features in nonsevere COVID-19 cases. Methods Data from nonsevere COVID-19 patients admitted to Haihe Hospital from May 2020 to June 2021 were retrospectively reviewed and analyzed. Results (1) A total of 122 cases were reviewed in the present study, including 38 mild and 84 moderate cases. The average age of mild cases was significantly different from moderate cases (P < 0.001). Eight patients complained of hyposmia and it was more frequent in mild cases (P < 0.001). The nucleic acid positive duration (NPD) of nonsevere novel coronavirus was 20.49 (confidence interval (CI) 17.50–3.49) days. (2) The levels of specific IgM and IgG for COVID-19 were higher in mild cases than in moderate cases (P=0.023 and P=0.047, respectively). (3) The correlation analysis with antibodies and T-cell subtypes showed that the lymphocyte (LYM) count, T cells, CD4+T cells, and CD8+T cells had a linear correlation with NPD. (4) Among the 93 patients monitored, 62 COVID-19 cases presented a progressive rise of specific IgM and IgG. The daily increase rates of IgM and IgG were 38.42% (CI 28.22–48.61%) and 24.90% (CI 0.23–29.58%), respectively. Conclusion The levels and daily increase rates of specific IgM and IgG against the virus can vary between cases. The NPD presented a linear correlation with the LYM, T cells, CD4+T cells, and CD8+T cells. Hence, more attention should be paid to these indicators in clinical practice.


Introduction
Te novel coronavirus disease 2019 (COVID- 19) is an acute respiratory infectious disease that comprehends a serious risk to human health [1,2] and has become a global pandemic. By November 1st, 2021, it has caused over 250 million infections and 5 million deaths worldwide [3]. At the molecular level, many parallels have been identifed between SARS and COVID-19, and the COVID-19 virus has been named SARS-CoV-2 [4]. Many studies regarding COVID- 19 have been performed and led to a certain understanding of its diagnosis, treatment, and prevention. COVID-19 patients can present diferent clinical symptoms including fever, fatigue, dry cough, myalgia, fu-like symptoms, and a very high mortality rate in severe and critical cases [5][6][7]. Some vaccines have been on the market and widely administered worldwide. However, there is still no efective drug to treat COVID-19. Hence, clinicians still rely only on the infected person's immune system to fght SARS-CoV-2.
Te components of the human immune system, such as lymphocytes (LYM), and the subsets of CD4 + T cells, CD8 + T cells, B cells, and natural killer (NK) cells, play an important role in the fght against the virus [8]. Additionally, immune disorders are an important factor in the development of severe COVID-19 [9]. In clinical practice, there are relatively few immune indicators for clinicians, including nonspecifc tests involving blood routine, lymphocyte subsets, and specifc immunoglobulin M (IgM) and immunoglobulin G (IgG) in serum. Recent studies have shown that lymphopenia (<20%) and severe lymphopenia (<5%) are observed in severe cases and CD8 + T cells can be a predictor of severe disease [9]. Moreover, virus-specifc IgM increases followed by virus-specifc IgGare detected during convalescence in the acute phase [10,11]. Te major discharge criteria are the novel coronavirus negative test on nasal and pharyngeal swabs. Since many nonsevere patients are selfquarantined at home once diagnosed in most countries, few studies have focused on the relationship between nucleic acid positive duration (NPD) of nonsevere novel coronavirus and immunological features in these cases. In China, all COVID-19 patients must be isolated and treated in designated hospitals. Terefore, we performed a comprehensive evaluation of the characteristics of 122 COVID-19 patients admitted to the designated hospital in Tianjin, China (Tianjin Haihe Hospital). Herein, we analyzed the clinical and immunological features of nonsevere cases. Our current fndings might help in the understanding of SARS-CoV-2 nucleic acid change in these cases, and the relationship between immunological indexes and COVID-19 prognoses.

Patients.
Patients with COVID•19 were recruited at the Tianjin Haihe hospital from May 2020 to June 2021. Te diagnostic criteria and clinical classifcation were based on the National Health Commission of China [12]. Tis was a retrospective study and no evidence that the enrolled patients were infected with a predominance of variants of concern was detected. After discharge, patients were kept under observation and health monitoring for 14 d.

Date Collection.
Demographic data (age, sex), epidemiological contact history, onset date, basic diseases, fever, hyposmia, blood routine (LYM), liver and kidney function, SARS-CoV-2 antibody, specifc IgM and IgG, T lymphocyte subsets, and other data were collected at the hospital. Te NPD was evaluated by throat or nose swabs and was defned as the time from the frst positive to the frst negative with a parallel controlled trial verifed by the local Disease Control and Prevention Center.

Statistical Analyses.
Te data were analyzed by SPSS 25.0 statistical and GraphPad Prism 8.0 software. Data are described as means, interquartile ranges, or percentages of the relative frequency according to the diference, and the 95% confdence interval (CI) was used. Te P values of comparisons between mild and moderate cases were derived from χ 2 , Fisher's exact, or unpaired two-sided Student's ttests. Furthermore, we performed a correlation analysis between the NPD, antibodies, and T-cell subtypes. A P < 0.05 was considered statistically signifcant.

Results
From May 2020 to June 2021, 166 COVID-19 cases were confrmed. According to the guidelines for diagnosis and management of COVID-19 (8th edition, in Chinese) issued by the National Health Commission of China [12], they were divided into mild, moderate, severe, and critical severe cases. Four severe cases and no critical severe cases were detected. Hence, 162 patients were diagnosed with nonsevere COVID-19. Furthermore, six patients with diabetes, one patient that received the vaccine, fve no adult cases, and 28 cases with insufcient data were excluded. Finally, the remaining 122 cases were enrolled in the present study, including 38 mild and 84 moderate cases. Te fowchart of the screening of nonsevere COVID-19 confrmed cases is presented in Figure 1.
Te average age of mild patients was 32.16 years and difered from moderate patients (39.50 years; P < 0.001). Six patients were Europeans and Americans and 116 were Asians. Fifteen patients were smokers. Fever was the most common symptom with a proportion of 23.77%. Eight patients complained of hyposmia and it was more frequent in mild cases (P < 0.001). Te NPD was 20.49 (17.50-3.49) days in all nonsevere COVID-19 cases, and the two groups did not difer. Te demographics and baseline characteristics of nonsevere COVID-19 patients are shown in Table 1.
Te laboratory tests of patients presented LYM of 1.94 (CI 1.76-2.11) × 10 9 /L, alanine aminotransferase (ALT) of 29.10 (CI 26.39-31.80) U/L, and Albumin (ALB) of 45.51 (CI 39.27-51.75) g/L. Te specifc IgM and IgG for COVID-19 were detected by chemiluminescence. Te lymphocyte subsets in the blood were analyzed by fow cytometry and the normal range of CD4 + T cell and the CD8 + T cell counts were 561 to 1137/MCL and 404 to 754/MCL, respectively. Te levels of specifc IgM and IgG for COVID-19 were higher in mild cases compared to moderate cases (P � 0.023 and P � 0.047, respectively). Te counts of CD4 + T and CD8 + T cells were in the normal range in most cases, but the count of mild patients was higher than moderate ones. Te immunological indexes of male and female nonsevere COVID-19 patients did not difer (Table 2).
Te correlation analysis for specifc antibodies and T cell subtypes showed that LYM, T cells, CD4 + T cells, and CD8 + T cells had a linear correlation with the NPD (Table 3, Figure 2). Additionally, 93 patients were monitored during their treatments and 62 COVID-19 cases presented a progressive rising trend of IgM and IgG levels. Te daily increase rate of IgM and IgG was 38.42% (CI 28.22-48.61%) and 24.90% (CI 0.23-29.58%), respectively. Te analysis of T-cell subtypes from 20 nonsevere COVID-19 patients showed that the count of T cells, CD4 + T cells, and CD8 + T cells did not difer between admission and discharge ( Table 4).

Discussion
Te COVID-19 infectious cases can be divided into mild, moderate, severe, and critical severe cases according to the guidelines for diagnosis and management of COVID-19 (8th   edition, in Chinese) issued by the National Health Commission of China [12]. Mild and moderate cases are considered nonsevere with respiratory symptoms of fever, imaging manifestations of pneumonia, no progressive dyspnea, and oxygenation index greater than 300. At the early stage of the COVID-19 pandemic, the investigation of 132 patients from the Wuhan Fourth Hospital who had COVID-19 from February 1 to 29 showed that the proportion of nonsevere cases was over 68.19% [13]. Herein, four cases were evaluated as severe or critical cases, and the remaining 162 (97.60%) were mild or moderate cases. Te reason for this phenomenon might be that the patients admitted were imported from other countries and most of them were healthy adults in the past. Diferent factors can afect the human immune status. Te immune system becomes mature at about 20 years in humans, and the immune system function progressively declines after 60 years [14]. Some underlying diseases, such as diabetes, can also afect the function of the immune system. Additionally, the metabolic alterations of patients sufering from obesity and diabetes mellitus (DM) can further afect the diferentiation, function, and survival of components of the innate and adaptive immunities [15,16]. COVID-19 patients with DM or obesity are at higher risk of death and are characterized by a state of chronic and lowgrade infammation because of the impaired innate and adaptive immune responses [17]. COVID-19 candidate vaccines induce a highly potent SARS-CoV-2 neutralizing antibody response [18] and elicit human antibody and TH1 T cell responses [19]. Terefore, we excluded six patients with diabetes, one patient that received the vaccine, fve patients under 18, and 28 cases with inefcient data. Finally, a total of 122 patients were enrolled in the present study, the age range was 20-59 years, and included 38 mild and 84 moderate patients.
In these cases, mild COVID-19 patients were younger than moderate ones (P < 0.001). Cough and fever were the most common respiratory symptoms, but hyposmia was detected in 23.68% of mild and 1.09% of moderate cases (P < 0.001). Te LYM and CD8 + T cell counts were statistically diferent between the two groups, consistent with previous studies [20,21]. Similar to many other viral infections, an increase in specifc IgM in the acute phase followed by an increase in specifc IgG at later phases has been observed in the course of COVID-19 [14]. An early increase in IgM followed by the development of IgG is a normal expected antibody response [9]. In the present study, the daily increase rates of IgM and IgG were also calculated    However, our study also has some limitations. First, we did not observe the dynamic changes of specifc antibodies and T-cell subtypes. Second, the postdischarge immune status of COVID-19 patients was not further assessed in the follow-up. Tird, some immunological indicators, including cytokines, were not observed due to limited conditions.
In summary, the identifcation of specifc IgM and IgG for COVID-19 is necessary, but their levels and daily increase rates might vary between cases. Moreover, the NPD presented a linear correlation with LYM, T cells, CD4 + T cells, and CD8 + T cells. Hence, more attention should be paid to these indexes in the clinic. Our current fndings provided a deeper insight into the disease's pathogenesis and might help in the precise therapeutic strategy for each COVID-19 patient.

Data Availability
Te datasets used and analyzed during this study are available from the corresponding author upon request.

Ethical Approval
Before its start, the study was approved by the ethics committee of Haihe Hospital, Tianjin, China. Te reference number for the study is 2020HHKT-026.

Disclosure
Dong Zhang, Xueren Li, and Haibai Sun are co-frst authors.