\documentclass[10pt]{article}

\usepackage{fullpage}
\usepackage{setspace}
\usepackage{parskip}
\usepackage{titlesec}
\usepackage[section]{placeins}
\usepackage{xcolor}
\usepackage{breakcites}
\usepackage{lineno}
\usepackage{hyphenat}





\PassOptionsToPackage{hyphens}{url}
\usepackage[colorlinks = true,
            linkcolor = blue,
            urlcolor  = blue,
            citecolor = blue,
            anchorcolor = blue]{hyperref}
\usepackage{etoolbox}
\makeatletter
\patchcmd\@combinedblfloats{\box\@outputbox}{\unvbox\@outputbox}{}{%
  \errmessage{\noexpand\@combinedblfloats could not be patched}%
}%
\makeatother


\usepackage{natbib}




\renewenvironment{abstract}
  {{\bfseries\noindent{\abstractname}\par\nobreak}\footnotesize}
  {\bigskip}

\titlespacing{\section}{0pt}{*3}{*1}
\titlespacing{\subsection}{0pt}{*2}{*0.5}
\titlespacing{\subsubsection}{0pt}{*1.5}{0pt}


\usepackage{authblk}


\usepackage{graphicx}
\usepackage[space]{grffile}
\usepackage{latexsym}
\usepackage{textcomp}
\usepackage{longtable}
\usepackage{tabulary}
\usepackage{booktabs,array,multirow}
\usepackage{amsfonts,amsmath,amssymb}
\providecommand\citet{\cite}
\providecommand\citep{\cite}
\providecommand\citealt{\cite}
% You can conditionalize code for latexml or normal latex using this.
\newif\iflatexml\latexmlfalse
\providecommand{\tightlist}{\setlength{\itemsep}{0pt}\setlength{\parskip}{0pt}}%

\AtBeginDocument{\DeclareGraphicsExtensions{.pdf,.PDF,.eps,.EPS,.png,.PNG,.tif,.TIF,.jpg,.JPG,.jpeg,.JPEG}}

\usepackage[utf8]{inputenc}
\usepackage[ngerman,greek,english]{babel}



\usepackage{float}








\begin{document}

\title{Effects of Coronavirus Disease 2019 (COVID-19) on peripheral blood
lymphocytes and their subsets in children: imbalanced CD4+/CD8 + T cell
ratio and disease severity}



\author[1]{Shima Mahmoudi}%
\author[1]{Bahareh  Yaghmaei}%
\author[1]{Meisam  Sharifzadeh Ekbatani}%
\author[1]{Babak  Pourakbari}%
\author[1]{Amene  Navaeian}%
\author[1]{Nima  Parvaneh}%
\author[1]{Mohammad Taghi  Haghi Ashtiani}%
\author[1]{Setareh Mamishi}%
\affil[1]{Affiliation not available}%


\vspace{-1em}



  
  \date{May 26, 2024}


\begingroup
\let\center\flushleft
\let\endcenter\endflushleft
\maketitle
\endgroup





\selectlanguage{english}
\begin{abstract}
Although the pathogenesis of Coronavirus Disease 2019 (COVID-19) is not
fully described, the interaction between the SARS-CoV-2 and the immune
system is so complicated, and lymphopenia, hyper-inflammatory responses,
and cytokines play an essential role in the pathology of COVID-19. Among
the laboratory features of COVID-19, some hematological abnormalities
have been described; however, no studies about lymphocyte subset
analyses have been reported in children. Therefore, this study aimed to
explain the characteristics of lymphocyte subsets in pediatrics with
mild or severe COVID-19. Subjects of the study were children with
confirmed severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)
pneumonia by real-time RT-PCR admitted to the Children's Medical Center,
affiliated to the Tehran University of Medical Sciences. Complete blood
count and lymphocyte subpopulations were studied for all patients. The
study population included 55 hospitalized patients with confirmed
SARS-CoV-2 infection (34 patients (62\%) with mild and 21 patients
(38\%) with severe disease). Lymphocyte counts were significantly lower
in patients with severe disease (mean\selectlanguage{ngerman}± sd 1.6± 0.9 in the severe group
vs. 2.3± 2.2 in the mild group). Compared with mild pneumonia, children
with severe pneumonia had a higher count of CD8+ lymphocyte, while a
lower percentage of CD4+ lymphocyte was observed; however, the
differences were not significant. Interestingly, severe group had a
significantly lower CD4+/CD8+ T cell ratio compared to the mild group
(1.1 ± 0.47 vs. 1.4 ± 0.8, p-value: 0.039). CD4+/CD8+ less than 2, 1.5,
and 1 was found in 48 (87\%), 40 (73\%), and 19 cases (35\%). Among 7
cases (13\%) who died, 100\% had CD4+/CD8+ ratio\textless{}2, 86\% had
CD4+/CD8+ ratio\textless{}1.5, and 29\% had CD4+/CD8 +
ratio\textless{}1. Lymphocyte counts and lymphocyte subset (CD4+ /CD8+ T
cell ratio) reflect the disease severity, which can be considered as a
potential predictor for disease severity of COVID-19 in children.%
\end{abstract}\selectlanguage{ngerman}%



\sloppy


\textbf{Introduction}

The Coronavirus Disease 2019 (COVID-19) caused by severe acute
respiratory syndrome corona virus 2 (SARS-CoV-2) has rapidly spread in
China and around the world and is still on-going (Sohrabi, Alsafi et al.
2020). All people, including children are susceptible to SARS-CoV-2, and
a rapid progression in morbidity and mortality rate due to
human-to-human transmission of the virus are still reported (Lotfi and
Rezaei 2020).

Although the pathogenesis of COVID-19 is not fully described, the
interaction between the SARS-CoV-2 and the immune system is so
complicated, and lymphopenia, hyper-inflammatory responses, and
cytokines play an important role in the pathogenesis of COVID-19
(Saghazadeh and Rezaei 2020, Yazdanpanah, Hamblin et al. 2020).

Recent studies suggest that the SARS-CoV-2 infection may primarily
affect T lymphocytes, particularly CD4\textsuperscript{+}~T and
CD8\textsuperscript{+}~T cells, which might be highly involved in the
pathological process of COVID-19 (Henry, de Oliveira et al. 2020, Wang,
Hu et al. 2020).

CD4\textsuperscript{+}~and~CD8\textsuperscript{+}~T~cells~play~a~fundamental~role~in~controlling~viral~infections~maintaining
cellular~and~humoral~immune~responses. These cells are involved in
humoral and cytotoxic immunity against viral infections.
CD4\textsuperscript{+}~T~cells~have multiple roles and are~required~to
support CD8\textsuperscript{+} T cell responses. Moreover, these cells
help to B cells to elicit antibody responses (Ng, Snell et al. 2013).
CD8\textsuperscript{+}~T cells have been shown to play a critical role
in mediating viral clearance after acute viral respiratory infections,
including respiratory syncytial virus, influenza A virus, and human
metapneumovirus (Wang, Nie et al. 2020).

Among the laboratory features of COVID-19, a number of hematological
abnormalities have been described (Henry, de Oliveira et al. 2020);
however, no studies about lymphocyte subset analyses have been reported
in children. Therefore, it is important to explain the characteristics
of lymphocyte subsets in pediatrics with mild or severe COVID-19. This
study aimed to evaluate the lymphocyte subsets in 55
laboratory-confirmed cases of SARS-CoV-2.

\textbf{Material and methods}

This study received ethical approval from the Tehran University of
Medical Sciences, Tehran, Iran. All participants gave written informed
consent, and the study was carried out following the Declaration of
Helsinki.

\subsubsection*{Patient selection}

{\label{patient-selection}}

Subjects of the study were children with confirmed SARS-CoV-2 pneumonia
by real-time RT-PCR (admitted between 07 March and 10 June 2020) in
Children's Medical Center, an Iranian referral hospital who underwent
the detection of peripheral blood lymphocyte subsets. The following
information on each patient was extracted from electronic medical
records: age, sex, underlying disease, intensive care unit (ICU)
admission, need for invasive mechanical ventilation, laboratory
findings, chest computed tomography (CT), and mortality. Chest CT scan
was reported by an expert radiologist, and abnormal CT finding was
considered if typical CT imaging features for COVID-19 including
peripheral, bilateral, ground-glass opacification with or without
consolidation or visible intralobular lines, multifocal ground-glass
opacification or rounded morphology with or without consolidation or
visible intralobular lines, or reverse halo sign or other findings of
organizing pneumonia were reported (Simpson, Kay et al. 2020). On
admission, severe illness was defined according to the following
criteria: (1) breathing rate [?]30 times/min; (2) pulse oximeter oxygen
saturation (SpO\textsubscript{2}) [?]93\% at rest; and (3) ratio of the
partial pressure of arterial oxygen (PaO\textsubscript{2}) to the
fraction of inspired oxygen (FiO\textsubscript{2}) [?]300 mmHg (Wang, Nie
et al. 2020).

\textbf{Real-time RT-PCR for SARS-CoV-2}

In all cases, nasopharyngeal samples were collected and tested for
SARS-CoV-2 with the LightMix\selectlanguage{ngerman}® Modular Wuhan CoV RdRP-gene Kit
(53-0777-96) by TIB MOLBIOL, Berlin, Germany (https://www.roche-as.es).
The rRT-PCR assay was performed according to the manufacturer's protocol
using probe P1 from the WHO protocol (RdRP\_SARSr-P1:
FAM-CCAGGTGGWACRTCATCMGGTGATGC-BBQ). The rRT-PCR assay was performed
following recommended cycling conditions: reverse transcription at 55 °C
for 5 min and 95 °C for 5 min, followed by 45 cycles of PCR at 95 °C for
15 sec and 60 °C for 15 sec. The cycle threshold (Ct) value above 39.0
was considered negative. The tested sample was considered SARS-CoV-2
positive if LightMix showed positive results for the RdRP gene.

\subsubsection*{Flow Cytometry for determination of lymphocyte
subsets}

{\label{flow-cytometry-for-determination-of-lymphocyte-subsets}}

Lymphocyte subsets were detected by flow cytometry analysis. Samples of
EDTA anticoagulated peripheral blood (2 mL) were collected from patients
with COVID-19 before initiation of treatment. All samples were tested
within 6 hours of being obtained, and CD4\textsuperscript{+} and
CD8\textsuperscript{+}~T-cell counts (cells/\selectlanguage{greek}μ\selectlanguage{english}L) were measured by
multiple-color flow cytometry. The cells were analyzed on a BD FACS
Canto II flow cytometry system (BD Biosciences).

{\label{lymphocyte-subsets-were-detected-by-flow-cytometry-analysis.-samples-of-edta-anticoagulated-peripheral-blood-2-ml-were-collected-from-patients-with-covid-19-before-initiation-of-treatment.-all-samples-were-tested-within-6-hours-of-being-obtained-and-cd4-and-cd8t-cell-counts-cellsux3bcl-were-measured-by-multiple-color-flow-cytometry.-the-cells-were-analyzed-on-a-bd-facs-canto-ii-flow-cytometry-system-bd-biosciences.}}

\subsubsection*{}

{\label{section-1}}

\subsubsection*{Statistical analysis}

{\label{statistical-analysis}}

Statistical analysis was performed with the Statistical Package for the
Social Sciences (SPSS version 18.0, SPSS Inc., Chicago, IL, USA).

Categorical data were described as percentages and continuous data as
median with interquartile range (IQR). Normally distributed continuous
variables were presented as means with standard deviations (SD).
Comparison of the differences between the two groups was conducted using
the t-test, Chi-square test, or Mann--Whitney U test. Variables with a
two-tailed p-value \textless{} 0.05 were considered statistically
significant.

\textbf{Results}

\textbf{Baseline data}

The study population included 55 hospitalized patients with confirmed
SARS-CoV-2 infection. The age of patients ranged from 20 days to 14.0
years (6.9 \selectlanguage{ngerman}± 4.1). Thirty-four patients (17 males and 17 females) with a
mean age of 6.9 ± 3.9 years had mild infection, and 21 patients (17
males and 4 females) with a mean age of 7.0±4.5 years were included in
the severe group. The severe form of the disease was significantly
higher in males than females (81\% vs. 19\%, p value= 0.02).

Among all patients, 9 cases admitted to the ICU, and 7 of them had
severe pneumonia (33\% in severe group and 6\% in mild group, p
value=0.02). Three patients received invasive mechanical ventilation.
The presence of underlying diseases was significantly higher in cases
with severe disease than cases in mild group (67\% vs. 29\%, p value=
0.011). Abnormal CT finding was found in 89\% and 67\% of cases with
severe and mild disease, respectively (p value= 0.14). The mortality
was13\% (n=7), and 6 deaths (86\%) occurred among cases with severe
disease.

\textbf{Laboratory findings}

Inflammatory indicators; erythrocyte sedimentation rate (ESR) and
C-reactive protein (CRP) were abnormal in 17 (85\%), and 17 (80\%)
patients in severe and 25 (81\%), and 24 (71\%) patients in mild group.

In blood tests of cases with severe pneumonia, leukocytes, lymphocytes,
and neutrophils were below the normal range in 3 (14\%), 6 (29\%), and 2
(9\%) patients, and above the normal range in 3 (15\%), 0 (0\%), and 5
(24\%) patients, respectively.~In another group with mild pneumonia
elevated leukocytes, lymphocytes, and neutrophils counts were seen in 6
(18\%), 1 (3\%), and 7 (21\%) patients, while it was below the normal
range in 4 (12\%), 13 (38\%), and 3 (9\%) patients, respectively. There
were no significant differences in white blood cell (WBC) count, Red
Blood Cells (RBC), hemoglobin, platelet, and neutrophil count between
the two groups. Lymphocyte count was significantly lower in patients
with severe disease (mean± sd 1.6± 0.9 in the severe group vs. 2.3± 2.2
in the mild group) (Table1).

\textbf{Lymphocyte subsets in peripheral blood of patients}

According to the results of each index, CD4\textsuperscript{+} T and
CD8\textsuperscript{+} T cells were divided into below the normal
values, within the normal value and above the normal values. The
corresponding quantities and proportions were respectively calculated
based on age, and the results were shown in Table 2.

Compared with mild pneumonia children, patients with severe pneumonia
had a higher count of CD8\textsuperscript{+} lymphocyte, while a lower
percentage of CD4\textsuperscript{+} lymphocyte was observed. However,
there were no significant differences in the percentage of
CD4\textsuperscript{+} and CD8\textsuperscript{+}lymphocytes between the
two groups (p-value= 0.3 and p-value= 0.4, respectively) (Figure 1).

\subsubsection*{\texorpdfstring{CD4\textsuperscript{+}/CD8\textsuperscript{+}T
cell ratio and COVID-19
Severity}{CD4+/CD8+T cell ratio and COVID-19 Severity}}

{\label{cd4cd8-t-cell-ratio-and-covid-19-severity}}

Interestingly, the severe group had a significantly lower
CD4\textsuperscript{+}/CD8\textsuperscript{+} T cell ratio compared to
the mild group (1.1 ± 0.47 vs. 1.4 ± 0.8, p value: 0.039) (Figure 1).

CD4\textsuperscript{+}/CD8\textsuperscript{+} less than 2, 1.5, and 1
was found in 48 (87\%), 40 (73\%), and 19 cases (35\%) , respectively.
The frequency of an inverted
CD4\textsuperscript{+}/CD8\textsuperscript{+} ratio increased with age.
A ratio\textless{}1 was seen in 32\% of patients less than 5 years (n=6)
and in 68\% of 5- to 15-year-olds (n=13). Males were more likely to have
a decreased ratio than females (Table 3).

Among 7 cases who died, 100\% had
CD4\textsuperscript{+}/CD8\textsuperscript{+} ratio\textless{}2, 86\%
had CD4\textsuperscript{+}/CD8\textsuperscript{+} ratio\textless{}1.5,
and 29\% had
CD4\textsuperscript{+}/CD8\textsuperscript{+}ratio\textless{}1.

\textbf{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\%\selectlanguage{english}-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\textsuperscript{+}~T cells and
CD8\textsuperscript{+}~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\textsuperscript{+}~T cells
was documented in the severe group than mild cases. The
CD4\textsuperscript{+} lymphocytopenia was more prevalent in the severe
group than the mild group (38\% vs. 29\%). On the other hand, higher
percentage of CD8\textsuperscript{+}~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 \emph{et al.} (Bai, Liu et al.
2020), Among 25 asymptomatic cases, very mild cases, 23 cases had normal
CD8\textsuperscript{+} T lymphocyte counts, while increased
CD8\textsuperscript{+} T lymphocyte counts were mildly increased in two
cases (8\%).

Progressive loss of CD4\textsuperscript{+}~T cells and an increase in
CD8\textsuperscript{+}~T cells are consistent features of HIV-1
infection. It has been reported that CD8\textsuperscript{+}~T cells'
count may change slightly, while the CD4\textsuperscript{+}~T cells
count decline at inconstant rate. A progressive decline in
CD4\textsuperscript{+}~T cells, as well as a rise in
CD8\textsuperscript{+}~T cells, suggests a compensatory rise in
CD8\textsuperscript{+}~T cells in response to a decline in
CD4\textsuperscript{+}~T cells (Margolick, Munoz et al. 1995).

Although lymphocyte count, especially CD4\textsuperscript{+}, 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\textsuperscript{+}~T cells and CD8\textsuperscript{+}~T cells and
severity of disease were found. On the other hand, the
CD4\textsuperscript{+}/CD8\textsuperscript{+} 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\textsuperscript{+}~T cells,
and CD8\textsuperscript{+}~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\textsuperscript{+}~T cells, and CD8\textsuperscript{+}~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\textsuperscript{+}~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\textsuperscript{+}~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\textsuperscript{+}~T, cell
counts.

The normal CD4\textsuperscript{+}/CD8\textsuperscript{+} ratio in
healthy hosts is poorly defined. Ratios between 1.5 and 2.5 are
generally considered normal (McBride and Striker 2017). Low
CD4\textsuperscript{+}/CD8\textsuperscript{+} ratios mainly observe in
HIV. However, a low or inverted
CD4\textsuperscript{+}/CD8\textsuperscript{+} ratio is associated with
systemic lupus erythematosus, chronic inflammation, and cytomegalovirus
infection as well (Maeda 1999, McBride and Striker 2017).
CD8\textsuperscript{+} T cells represent an important immunological
component of protection against viral infections. Cytomegalovirus has a
significant impact on the
CD4\textsuperscript{+}/CD8\textsuperscript{+}ratio by increasing
circulating CD8\textsuperscript{+} cells in response to the infection
(Reker-Hadrup, Strindhall et al. 2006).

Inversion of the CD4\textsuperscript{+}/CD8\textsuperscript{+} 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\textsuperscript{+}/CD8\textsuperscript{+} ratios (\textless{}1.5).

A decrease in CD4\textsuperscript{+}/CD8\textsuperscript{+} ratio
indicates the extent of the impairment of the immune system by COVID-19.
Therefore, a decline in CD4\textsuperscript{+}/CD8\textsuperscript{+} 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\textsuperscript{+}/CD8\textsuperscript{+}~T~cell ratio can be
considered as a potential predictor for disease severity of COVID-19.

\textbf{Acknowledgments}

The authors would like to thank the patients who participated in this
study, and the staff at Children's Medical Center for their assistance
with study enrollment, data collection, and sample examination.

\textbf{Conflict of interests}

The authors declare that there is no conflict of interest.

\textbf{Availability of data:} The data that support the findings of
this study are available on request from the corresponding author. The
data are not publicly available due to privacy or ethical restrictions.

\textbf{References}

Bai, K., W. Liu, C. Liu, Y. Fu, J. Hu, Y. Qin, Q. Zhang, H. Chen, F. Xu
and C. Li (2020). ''Clinical Analysis of 25 COVID-19 Infections in
Children.'' Pediatr Infect Dis J\textbf{39} (7): e100-e103.

Bi, Q., Y. Wu, S. Mei, C. Ye, X. Zou, Z. Zhang, X. Liu, L. Wei, S. A.
Truelove and T. Zhang (2020). ''Epidemiology and transmission of
COVID-19 in 391 cases and 1286 of their close contacts in Shenzhen,
China: a retrospective cohort study.'' The Lancet Infectious Diseases.

Bitnun, A., U. Allen, H. Heurter, S. M. King, M. A. Opavsky, E. L.
Ford-Jones, A. Matlow, I. Kitai, R. Tellier and S. Richardson (2003).
''Children hospitalized with severe acute respiratory syndrome-related
illness in Toronto.'' Pediatrics\textbf{112} (4): e261-e261.

Dong, Y., X. Mo, Y. Hu, X. Qi, F. Jiang, Z. Jiang and S. Tong (2020).
''Epidemiological characteristics of 2143 pediatric patients with 2019
coronavirus disease in China.'' Pediatrics.

Guan, W.-j., Z.-y. Ni, Y. Hu, W.-h. Liang, C.-q. Ou, J.-x. He, L. Liu,
H. Shan, C.-l. Lei and D. S. Hui (2020). ''Clinical characteristics of
coronavirus disease 2019 in China.'' New England Journal of Medicine.

Guo, Y.-R., Q.-D. Cao, Z.-S. Hong, Y.-Y. Tan, S.-D. Chen, H.-J. Jin,
K.-S. Tan, D.-Y. Wang and Y. Yan (2020). ''The origin, transmission and
clinical therapies on coronavirus disease 2019 (COVID-19) outbreak--an
update on the status.'' Military Medical Research \textbf{7} (1): 1-10.

Henry, B. M. (2020). ''COVID-19, ECMO, and lymphopenia: a word of
caution.'' The Lancet Respiratory Medicine\textbf{8} (4): e24.

Henry, B. M., M. H. S. de Oliveira, S. Benoit, M. Plebani and G. Lippi
(2020). ''Hematologic, biochemical and immune biomarker abnormalities
associated with severe illness and mortality in coronavirus disease 2019
(COVID-19): a meta-analysis.'' Clin Chem Lab Med.

Hon, K., C. Leung, W. Cheng, P. Chan, W. Chu, Y. Kwan, A. Li, N. Fong,
P. Ng and M. Chiu (2003). ''Clinical presentations and outcome of severe
acute respiratory syndrome in children.'' The Lancet \textbf{361}
(9370): 1701-1703.

Huppert, F. A., E. M. Pinto, K. Morgan, M. CFAS and C. Brayne (2003).
''Survival in a population sample is predicted by proportions of
lymphocyte subsets.'' Mechanisms of ageing and development \textbf{124}
(4): 449-451.

Liu, J., S. Li, B. Liang, X. Wang, H. Wang, W. Li, Q. Tong, J. Yi, L.
Zhao, L. Xiong, C. Guo, J. Tian, J. Luo, J. Yao, R. Pang, H. Shen, C.
Peng, T. Liu, Q. Zhang, J. Wu, L. Xu, S. Lu, B. Wang, Z. Weng, C. Han,
H. Zhu, R. Zhou, H. Zhou, X. Chen, P. Ye, B. Zhu, L. Wang, W. Zhou, S.
He, Y. He, S. Jie, P. Wei, J. Zhang, Y. Lu, W. Wang, L. Zhang, L. Li, F.
Zhou, J. Wang, U. Dittmer, M. Lu, Y. Hu, D. Yang and X. Zheng (2020).
''Longitudinal characteristics of lymphocyte responses and cytokine
profiles in the peripheral blood of SARS-CoV-2 infected patients.''
EBioMedicine \textbf{55} : 102763.

Lotfi, M. and N. Rezaei (2020). ''SARS-CoV-2: A comprehensive review
from pathogenicity of the virus to clinical consequences.'' J Med Virol.

Maeda, I. S., N Iida, M Matsumoto, H Hashimoto, S Hirose, N (1999).
''Relationship between CD4+/CD8+ T cell ratio and T cell activation in
systemic lupus erythematosus.'' Scandinavian journal of rheumatology
\textbf{28} (3): 166-170.

Margolick, J. B., A. Munoz, A. D. Donnenberg, L. P. Park, N. Galai, J.
V. Giorgi, M. R. O'Gorman and J. Ferbas (1995). ''Failure of T-cell
homeostasis preceding AIDS in HIV-1 infection. The Multicenter AIDS
Cohort Study.'' Nat Med \textbf{1} (7): 674-680.

McBride, J. A. and R. Striker (2017). ''Imbalance in the game of T
cells: what can the CD4/CD8 T-cell ratio tell us about HIV and health?''
PLoS pathogens \textbf{13} (11).

Ng, C. T., L. M. Snell, D. G. Brooks and M. B. Oldstone (2013).
''Networking at the level of host immunity: immune cell interactions
during persistent viral infections.'' Cell Host Microbe \textbf{13} (6):
652-664.

Ni, M., F. B. Tian, D. D. Xiang and B. Yu (2020). ''Characteristics of
inflammatory factors and lymphocyte subsets in patients with severe
COVID-19.'' J Med Virol.

Reker-Hadrup, S., J. Strindhall, T. Kollgaard, T. Seremet, B. Johansson,
G. Pawelec, P. Strat and A. Wikby (2006). ''Longitudinal studies of
clonally expanded CD8 T cells reveal a repertoire shrinkage predicting
mortality and increased number of dysfunctional cytomegalovirus-specific
T cells in the elderly.'' J Immunol \textbf{176} (4): 2645-2653.

Saghazadeh, A. and N. Rezaei (2020). ''Immune-epidemiological parameters
of the novel coronavirus--a perspective.'' Expert Review of Clinical
Immunology: 1-6.

Simpson, S., F. U. Kay, S. Abbara, S. Bhalla, J. H. Chung, M. Chung, T.
S. Henry, J. P. Kanne, S. Kligerman and J. P. Ko (2020). ''Radiological
Society of North America Expert Consensus Statement on Reporting Chest
CT Findings Related to COVID-19. Endorsed by the Society of Thoracic
Radiology, the American College of Radiology, and RSNA.'' Radiology:
Cardiothoracic Imaging \textbf{2} (2): e200152.

Sohrabi, C., Z. Alsafi, N. O'Neill, M. Khan, A. Kerwan, A. Al-Jabir, C.
Iosifidis and R. Agha (2020). ''World Health Organization declares
global emergency: A review of the 2019 novel coronavirus (COVID-19).''
International Journal of Surgery.

Sun, D., H. Li, X.-X. Lu, H. Xiao, J. Ren, F.-R. Zhang and Z.-S. Liu
(2020). ''Clinical features of severe pediatric patients with
coronavirus disease 2019 in Wuhan: a single center's observational
study.'' World Journal of Pediatrics: 1-9.

Tang, X., C. Wu, X. Li, Y. Song, X. Yao, X. Wu, Y. Duan, H. Zhang, Y.
Wang and Z. Qian (2020). ''On the origin and continuing evolution of
SARS-CoV-2.'' National Science Review.

Wang, D., B. Hu, C. Hu, F. Zhu, X. Liu, J. Zhang, B. Wang, H. Xiang, Z.
Cheng and Y. Xiong (2020). Clinical characteristics of 138 hospitalized
patients with 2019 novel coronavirus-infected pneumonia in Wuhan, China.
JAMA {[}Internet{]}. 2020 Feb {[}cited 2020 Mar 23{]}; 323 (11): 1061-9.

Wang, F., J. Nie, H. Wang, Q. Zhao, Y. Xiong, L. Deng, S. Song, Z. Ma,
P. Mo and Y. Zhang (2020). ''Characteristics of Peripheral Lymphocyte
Subset Alteration in COVID-19 Pneumonia.'' J Infect Dis \textbf{221}
(11): 1762-1769.

Yang, A. P., H. M. Li, W. Q. Tao, X. J. Yang, M. Wang, W. J. Yang and J.
P. Liu (2020). ''Infection with SARS-CoV-2 causes abnormal laboratory
results of multiple organs in patients.'' Aging (Albany NY) \textbf{12}
.

Yazdanpanah, F., M. R. Hamblin and N. Rezaei (2020). ''The immune system
and COVID-19: Friend or foe?'' Life Sci: 117900.

Zhang, W., Y. Zhao, F. Zhang, Q. Wang, T. Li, Z. Liu, J. Wang, Y. Qin,
X. Zhang and X. Yan (2020). ''The use of anti-inflammatory drugs in the
treatment of people with severe coronavirus disease 2019 (COVID-19): The
experience of clinical immunologists from China.'' Clinical Immunology:
108393.

Table 1.~The patients' laboratory findings and blood lymphocyte subsets
of children with COVID-19\selectlanguage{english}
\begin{longtable}[]{@{}llll@{}}
\toprule
Parameter & Mild (Mean\selectlanguage{ngerman}± SD) & Severe (Mean\selectlanguage{ngerman}± SD) & P value\tabularnewline
\midrule
\endhead
White blood cell count (\selectlanguage{ngerman}× 10\selectlanguage{english}9 cells per L) & 8.8\selectlanguage{ngerman}± 6.1 & 8.6\selectlanguage{ngerman}± 4.3 &
0.4\tabularnewline
Neutrophil count (\selectlanguage{ngerman}× 10\selectlanguage{english}9 cells per L) & 5.8\selectlanguage{ngerman}± 5.0 & 6.2\selectlanguage{ngerman}± 3.9 &
0.69\tabularnewline
Lymphocyte count (\selectlanguage{ngerman}× 10\selectlanguage{english}9 cells per L) & 2.3\selectlanguage{ngerman}± 2.2 & 1.6\selectlanguage{ngerman}± 0.9 &
0.05\tabularnewline
Platelet count (\selectlanguage{ngerman}× 10\selectlanguage{english}9 cells per L) & 272.5\selectlanguage{ngerman}± 159 & 270\selectlanguage{ngerman}± 156 &
0.9\tabularnewline
Red Blood Cells (\selectlanguage{ngerman}× 10\selectlanguage{english}9 cells per L) & 4.4\selectlanguage{ngerman}± 0.7 & 4.4\selectlanguage{ngerman}± 0.1 &
0.19\tabularnewline
Hemoglobin (g.dL) & 11.9\selectlanguage{ngerman}± 1.9 & 11.1\selectlanguage{ngerman}± 2.6 & 0.32\tabularnewline
CD4\textsuperscript{+} T~cells \% & 34.8\selectlanguage{ngerman}± 11.2 & 32\selectlanguage{ngerman}± 8.6 &
0.9\tabularnewline
CD8\textsuperscript{+} T~cells \% & 27.7\selectlanguage{ngerman}± 10.1 & 31.3\selectlanguage{ngerman}± 8.1 &
0.9\tabularnewline
C-reactive protein (mg.L)* & 14 (4-62) & 34 (7.2-69) &
0.22\tabularnewline
Erythrocyte sedimentation rate (mm/h)* & 22 (14-41) & 40.5 (23.5-58) &
0.03\tabularnewline
\bottomrule
\end{longtable}

*Median (Interquartile range)

Table 2.~The blood lymphocyte subsets of children with COVID-19\selectlanguage{english}
\begin{longtable}[]{@{}llllllll@{}}
\toprule
COVID-19 Severity & & CD4\textsuperscript{+} T~cells &
CD4\textsuperscript{+} T~cells & CD4\textsuperscript{+} T~cells &
CD8\textsuperscript{+} T~cells & CD8\textsuperscript{+} T~cells &
CD8\textsuperscript{+} T~cells\tabularnewline
\midrule
\endhead
& & Below normal range & Within normal range & Above normal range &
Below normal range & Within normal range & Above normal
range\tabularnewline
Mild & N & 10 & 21 & 3 & 4 & 24 & 6\tabularnewline
& \% & 29 & 62 & 9 & 12 & 70 & 18\tabularnewline
Severe & N & 8 & 13 & 0 & 1 & 14 & 6\tabularnewline
& \% & 3 & 92 & 0 & 5 & 67 & 29\tabularnewline
Total & N & 18 & 34 & 3 & 5 & 38 & 12\tabularnewline
& \% & 33 & 62 & 5.5 & 9 & 69 & 22\tabularnewline
\bottomrule
\end{longtable}

Table 3. Demographic and laboratory findings of children with
COVID-19~according to different
CD4\textsuperscript{+}/CD8\textsuperscript{+} T cells ratio\selectlanguage{english}
\begin{longtable}[]{@{}llllllllll@{}}
\toprule
Parameter & & CD4\textsuperscript{+}/CD8\textsuperscript{+} T cells
ratio & CD4\textsuperscript{+}/CD8\textsuperscript{+} T cells ratio &
CD4\textsuperscript{+}/CD8\textsuperscript{+} T cells ratio &
CD4\textsuperscript{+}/CD8\textsuperscript{+} T cells ratio &
CD4\textsuperscript{+}/CD8\textsuperscript{+} T cells ratio &
CD4\textsuperscript{+}/CD8\textsuperscript{+} T cells ratio &
CD4\textsuperscript{+}/CD8\textsuperscript{+} T cells ratio &
CD4\textsuperscript{+}/CD8\textsuperscript{+} T cells
ratio\tabularnewline
\midrule
\endhead
& & \textless{}1 (N=19) & \textless{}1 (N=19) & \textless{}1.5 (N=40) &
\textless{}1.5 (N=40) & \textless{}2 (N=48) & \textless{}2 (N=48) & [?]2
(N=7) & [?]2 (N=7)\tabularnewline
& & N & \% & N & \% & N & \% & N & \%\tabularnewline
Mild & & 9 & 47 & 23 & 57.5 & 28 & 58 & 6 & 86\tabularnewline
Severe & & 10 & 53 & 17 & 42.5 & 20 & 42 & 1 & 14\tabularnewline
Sex & Male & 12 & 63 & 26 & 65 & 31 & 65 & 3 & 43\tabularnewline
& Female & 7 & 37 & 14 & 35 & 17 & 35 & 4 & 57\tabularnewline
Age & \textless{} 1 year & 1 & 5 & 2 & 5 & 2 & 4 & 2 & 29\tabularnewline
& 1 to 5 year & 5 & 26 & 12 & 30 & 17 & 35 & 3 & 43\tabularnewline
& 5 to 10 year & 6 & 32 & 15 & 37.5 & 16 & 33 & 2 & 29\tabularnewline
& 10 to 15 year & 7 & 37 & 10 & 25 & 12 & 25 & 0 & 0\tabularnewline
Death & & 2 & 11 & 6 & 15 & 7 & 15 & 0 & 0\tabularnewline
Underlying disease & & 10 & 53 & 20 & 50 & 23 & 48 & 1 &
14\tabularnewline
Abnormal CT findings & & 10 & 53 & 21 & 52.5 & 26 & 54 & 4 &
57\tabularnewline
ICU admission & & 4 & 21 & 8 & 20 & 8 & 17 & 1 & 14\tabularnewline
Invasive mechanical ventilation & & 2 & 11 & 3 & 7.5 & 3 & 6 & 0 &
0\tabularnewline
White blood cell count (\selectlanguage{ngerman}× 10\selectlanguage{english}9 cells per L) & Below normal range & 3 & 16
& 6 & 15 & 7 & 15 & 0 & 0\tabularnewline
& Within Normal range & 13 & 53 & 27 & 67.5 & 34 & 71 & 5 &
71\tabularnewline
& Above normal range & 3 & 16 & 7 & 17.5 & 7 & 15 & 2 &
29\tabularnewline
Neutrophil count (\selectlanguage{ngerman}× 10\selectlanguage{english}9 cells per L) & Below normal range & 3 & 16 & 5 &
12.5 & 5 & 10 & 0 & 0\tabularnewline
& Within Normal range & 11 & 58 & 25 & 32.5 & 33 & 69 & 5 &
71\tabularnewline
& Above normal range & 5 & 26 & 10 & 25 & 10 & 21 & 2 &
29\tabularnewline
Lymphocyte count (\selectlanguage{ngerman}× 10\selectlanguage{english}9 cells per L) & Below normal range & 6 & 32 & 14
& 35 & 16 & 33 & 3 & 43\tabularnewline
& Within Normal range & 13 & 53 & 25 & 32.5 & 31 & 62.5 & 4 &
57\tabularnewline
& Above normal range & 0 & 0 & 1 & 2.5 & 1 & 2 & 0 & 0\tabularnewline
Platelet count (\selectlanguage{ngerman}× 10\selectlanguage{english}9 cells per L) & Below normal range & 5 & 26 & 11 &
27.5 & 13 & 27 & 1 & 14\tabularnewline
& Within Normal range & 12 & 63 & 23 & 57.5 & 28 & 58 & 5 &
71\tabularnewline
& Above normal range & 2 & 11 & 6 & 15 & 7 & 15 & 1 & 14\tabularnewline
C-reactive protein (mg.L) & Above normal range & 15 & 79 & 30 & 75 & 36
& 75 & 5 & 71\tabularnewline
Erythrocyte sedimentation rate (mm/h) & Above normal range & 14 & 74 &
30 & 75 & 37 & 77 & 5 & 71\tabularnewline
\bottomrule
\end{longtable}

\textbf{Hosted file}

\verb`image1.emf`  available at \url{https://authorea.com/users/339735/articles/465883-effects-of-coronavirus-disease-2019-covid-19-on-peripheral-blood-lymphocytes-and-their-subsets-in-children-imbalanced-cd4-cd8-t-cell-ratio-and-disease-severity}

\textbf{Hosted file}

\verb`image2.emf`  available at \url{https://authorea.com/users/339735/articles/465883-effects-of-coronavirus-disease-2019-covid-19-on-peripheral-blood-lymphocytes-and-their-subsets-in-children-imbalanced-cd4-cd8-t-cell-ratio-and-disease-severity}

P value= 0.3 P value= 0.4 P value= 0.039

\textbf{Hosted file}

\verb`image3.emf`  available at \url{https://authorea.com/users/339735/articles/465883-effects-of-coronavirus-disease-2019-covid-19-on-peripheral-blood-lymphocytes-and-their-subsets-in-children-imbalanced-cd4-cd8-t-cell-ratio-and-disease-severity}

Figure 1.~Profile of CD4\textsuperscript{+} T cells,
CD8\textsuperscript{+} T cells and
CD4\textsuperscript{+}/CD8\textsuperscript{+} T cells ratio in
lymphocytes in children with COVID-19

\selectlanguage{english}
\FloatBarrier
\end{document}

