Discussion
To our knowledge, this is the first preliminary study evaluating the lymphocyte subset characteristics of children with laboratory-confirmed SARS-CoV-2 infection.
Although early studies reported that children were less likely to develop COVID-19 than other age groups, recent studies revealed that children are at a similar risk of infection to the general population (Bi, Wu et al. 2020, Lotfi and Rezaei 2020). Consistent with previous reports (Dong, Mo et al. 2020, Guan, Ni et al. 2020, Sun, Li et al. 2020, Tang, Wu et al. 2020), the present study showed a male predominance in the incidence of COVID-19.
Changes in the immune system may result in decreased immune function and increased incidence of severity, morbidity, and mortality of infections (1, 2). According to the laboratory findings, normal leukocytes counts were more common in both cases with severe and mild disease that was similar to the previous report in children (Bai, Liu et al. 2020). It has been hypothesized that the repletion of lymphocytes plays a vital role in the recovery of patients with COVID-19 (Henry 2020). Lymphopenia and inflammatory cytokine storm during highly pathogenic coronavirus infections, such as SARS coronavirus (SARS-CoV), MERS coronavirus (MERS-CoV), and SARS COV-2 infections might be associated with disease severities (Yang, Li et al. 2020).
Although 63%−84.6% of adult patients with severe diseases have lymphopenia (Guo, Cao et al. 2020, Liu, Li et al. 2020), in our study, lymphopenia was found in 19 children (34.5%). Lymphocyte count was significantly lower in patients with severe disease. So, a significant decrease in lymphocytes may signify clinical worsening and increased risk of a poor outcome. According to previous reports in children with probable or suspected SARS (Bitnun, Allen et al. 2003, Hon, Leung et al. 2003), total lymphopenia was common and more prominent in older children with more severe disease. The counts of total leukocytes, RBCs, platelets, and neutrophils were not significantly different in severe and mild groups.
Inflammatory markers, including CRP and ESR, were higher in severe cases than in mild cases, demonstrating a higher inflammatory state during severe infection.  Since cytokine storm is associated with apoptosis of lymphocytes, a decrease in number of peripheral lymphocytes in severe cases is probable (Ni, Tian et al. 2020).
Although an apparent decrease in peripheral lymphocytes in COVID-19 patients has been reported, alteration in the T cells subsets is still unclear (Wang, Nie et al. 2020, Zhang, Zhao et al. 2020). However, it has been reported that CD4+ T cells and CD8+ T cells decreased in severe cases of COVID-19 than mild cases (Liu, Li et al. 2020, Ni, Tian et al. 2020, Wang, Nie et al. 2020). In our study higher number of CD8+ T cells was documented in the severe group than mild cases. The CD4+ lymphocytopenia was more prevalent in the severe group than the mild group (38% vs. 29%). On the other hand, higher percentage of CD8+ T cells was found in the severe group (29%) than in the mild group (18%); however, these differences were not significant. In the study of Bai et al. (Bai, Liu et al. 2020), Among 25 asymptomatic cases, very mild cases, 23 cases had normal CD8+ T lymphocyte counts, while increased CD8+ T lymphocyte counts were mildly increased in two cases (8%).
Progressive loss of CD4+ T cells and an increase in CD8+ T cells are consistent features of HIV-1 infection. It has been reported that CD8+ T cells’ count may change slightly, while the CD4+ T cells count decline at inconstant rate. A progressive decline in CD4+ T cells, as well as a rise in CD8+ T cells, suggests a compensatory rise in CD8+ T cells in response to a decline in CD4+ T cells (Margolick, Munoz et al. 1995).
Although lymphocyte count, especially CD4+, has been reported as a clinical predictor of severity of disease (Henry, de Oliveira et al. 2020), in our study, no significant differences between CD4+ T cells and CD8+ T cells and severity of disease were found. On the other hand, the CD4+/CD8+ T cell ratio was significantly lower in those patients with severe disease compared to those with mild disease. In contrast to previous reports in adults that absolute numbers of total T lymphocytes, CD4+ T cells, and CD8+ T cells were reduced below the lower limit of normal in the vast majority of patients with either severe or moderate COVID-19 (Wang, Hu et al. 2020), we demonstrated that percentage of CD4+ T cells, and CD8+ T cells were reduced below the lower limit of normal in 29% and 38% of cases with mild and severe COVID-19, respectively. However, CD8+ T cells were reduced below the lower limit of normal in 12% and 5% of cases with mild and severe COVID-19, respectively.
The low frequency of lymphopenia in severe patients is mainly related to the significantly decreased absolute counts of T cells, especially CD8+ T cells (Liu, Li et al. 2020), therefore; low rate of lymphopenia in children might be due to the fact that a majority of children have normal or even elevated CD8+ T, cell counts.
The normal CD4+/CD8+ ratio in healthy hosts is poorly defined. Ratios between 1.5 and 2.5 are generally considered normal (McBride and Striker 2017). Low CD4+/CD8+ ratios mainly observe in HIV. However, a low or inverted CD4+/CD8+ ratio is associated with systemic lupus erythematosus, chronic inflammation, and cytomegalovirus infection as well (Maeda 1999, McBride and Striker 2017). CD8+ T cells represent an important immunological component of protection against viral infections. Cytomegalovirus has a significant impact on the CD4+/CD8+ratio by increasing circulating CD8+ cells in response to the infection (Reker-Hadrup, Strindhall et al. 2006).
Inversion of the CD4+/CD8+ ratios are associated with poor survival (Huppert, Pinto et al. 2003), a link between low ratio and poor outcomes observed in our study, and importantly 86% of death rates (n=6) was reported in cases with low CD4+/CD8+ ratios (<1.5).
A decrease in CD4+/CD8+ ratio indicates the extent of the impairment of the immune system by COVID-19. Therefore, a decline in CD4+/CD8+ 8 ratio may serve as a useful factor to reflect the derangement of immune responses and even death in patients with severe COVID-19.
In conclusion, lymphocyte counts and inverted CD4+/CD8+ T cell ratio can be considered as a potential predictor for disease severity of COVID-19.