\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,english]{babel}



\usepackage{float}








\begin{document}

\title{COVID-19 in an adolescent with aplastic anemia undergoing
immunosuppressive therapy: a case report and details of antibody testing
for SARS-CoV-2}



\author[1]{Shota Kato}%
\author[1]{Yoshiko Nakano}%
\author[1]{Yuki Nakano}%
\author[1]{Koh Okamoto}%
\author[1]{Nao Takasugi}%
\author[1]{Moe Hidaka}%
\author[1]{Masahiro Sekiguchi}%
\author[1]{Mitsuteru Hiwatari}%
\author[1]{Makoto Kurano}%
\author[1]{Motohiro Kato}%
\affil[1]{The University of Tokyo}%


\vspace{-1em}



  
  \date{May 22, 2024}


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





\selectlanguage{english}
\begin{abstract}
The clinical course of COVID-19 in pediatric patients with aplastic
anemia (AA) have not been thoroughly investigated. We report a case of
COVID-19 in a 15-year-old male with AA treated via immunosuppressive
therapy. The patient initially presented with a fever and mild sore
throat. He continued cyclosporine treatment, and his symptoms improved
with a single dose of hydrocortisone. Antibody testing showed increased
anti-S1 and anti-RBD IgA levels, followed by elevation of anti-S1 and
anti-N IgM/IgG levels. Our results suggest that AA is not necessarily
associated with a higher risk of severe COVID-19.%
\end{abstract}%



\sloppy


\textbf{INTRODUCTION}

The increase in the cumulative incidence of COVID-19 cases was
accompanied by a gradual increase in reported pediatric
cases.\textsuperscript{1} However, the clinical course of COVID-19 in
pediatric patients with aplastic anemia (AA) has not been thoroughly
investigated. As of May 2021, only one pediatric and eight adult cases
of COVID-19 in patients with AA have been reported. Some patients
exhibited mild and transient symptoms, while others suffered from severe
manifestations with fatal outcomes.\textsuperscript{2-6} Data on the
ability of these patients to produce antibodies against the virus under
insufficient immune function are also limited.

We report our experience with COVID-19 in a patient with AA receiving
immunosuppressive therapy (IST) and the dynamic results of the
serological assay. We analyzed the change in immunoglobulin G (IgG),
immunoglobulin M (IgM), and immunoglobulin A (IgA) titers against three
different proteins of severe acute respiratory syndrome coronavirus 2
(SARS-CoV-2): S1 subunit of spike protein (S1); receptor-binding domain
(RBD) within the S1 subunit, which is a major target of anti-SARS-CoV-2
neutralizing antibodies\textsuperscript{7-9}; and nucleocapsid protein
(N).

\textbf{CASE DESCRIPTION}

A 15-year-old male receiving cyclosporine for AA was admitted to the
University of Tokyo Hospital due to fever. He was diagnosed with severe
AA four months ago. Since a human leukocyte antigen-matched sibling
donor was unavailable, he received IST with corticosteroids, rabbit
antithymocyte globulin, and cyclosporine combined with eltrombopag. He
was discharged and barely achieved a partial response two months after
IST initiation (Fig. 1). At the time of his hospitalization, he
presented with a fever (39.2@\selectlanguage{english}C) and mild sore throat that was noted one
day previously. His white blood cell count was 1.0 \selectlanguage{ngerman}×
10\textsuperscript{9}/L, with absolute neutrophil and lymphocyte counts
of 0.67 × 10\textsuperscript{9}/L and 0.18 × 10\textsuperscript{9}/L,
respectively. The hemoglobin level was 9.8 g/dL, and the platelet count
was 45 × 10\textsuperscript{9}/L without transfusion for more than two
months. The serum IgG level was low at 503 mg/dL (reference value,
861--1747 mg/dL). Other blood examinations were unremarkable. The chest
radiograph did not show any evidence of pneumonia.

On day 2 (the onset of COVID-19 was considered day 0), the patient
tested positive on nasopharyngeal swab polymerase chain reaction testing
for SARS-CoV-2. As a part of IST, cyclosporine was continued with trough
concentrations of 150--250 ng/mL. After receiving intravenous
immunoglobulin and a single dose of hydrocortisone, he became afebrile
on day 2. Although the complete blood count fluctuated slightly, he did
not require transfusion or granulocyte-colony stimulating factor. He was
discharged on day 10 without sequelae attributed to COVID-19 for four
months.

\textbf{IMMUNOLOGICAL ASSAY}

To investigate antibody kinetics to SARS-CoV-2, we performed serial
SARS-CoV-2 serological tests by chemiluminescent immunoassay using
iFlash 3000 and iFlash-SARS-CoV-2 IgG/IgM/IgA kits (Shenzhen YHLO
Biotech Co., Ltd., Shenzhen, China). The serum IgG, IgM, and IgA
antibodies against S1, RBD, and N were quantified. The patient's blood
samples collected on days -8 (during a regular checkup), 1, 2, 4, 6, 9,
32, 59, 79, 100, and 136 were examined. The cutoff values used were
based on the results of 249 samples collected from the University of
Tokyo Hospital (137 samples from SARS-CoV-2 RNA-positive patients and
112 samples from SARS-CoV-2 RNA-negative patients).

The anti-S1 and anti-RBD IgA levels exceeded the cutoff levels on day 9
and rapidly decreased thereafter. The anti-N IgM level was elevated on
day 32. The anti-S1 IgG and IgM and the anti-N IgG levels also
increased, but their titers did not exceed the cutoff values. Meanwhile,
the anti-RBD IgG and IgM levels hardly changed (Fig. 2).

\textbf{DISCUSSION}

Our patient exhibited no severe symptoms and fully recovered from
COVID-19 despite his immunosuppressed status. The clinical course of
COVID-19 was reportedly less severe in the pediatric population than
adults.\textsuperscript{1} His young age was possibly a contributing
factor for the mild clinical course. Other known risk factors for severe
COVID-19 include underlying diseases such as hypertension, diabetes,
obesity, and lung diseases. However, the relationship between
immunocompromised states due to the underlying disease or
immunosuppressive treatment and COVID-19 severity remains controversial.
Some studies reported that immunocompromised patients had favorable
outcomes compared to the general
population.\textsuperscript{10,11}Hyperactivation of immune response and
excessive inflammatory reaction were associated with the pathogenesis of
severe COVID-19.\textsuperscript{12} Among the previously reported nine
cases of COVID-19 in patients with AA, there was one patient who
developed COVID-19 during cyclosporine treatment and the patient fully
recovered.\textsuperscript{3} Therefore, immunosuppression from AA and
IST, including continued cyclosporine use, possibly contributed to the
uncomplicated course in our patient, despite an increased risk of viral
invasion and delayed viral elimination.

To evaluate the immune response to SARS-CoV-2 in this patient, a
serological assay was conducted. The anti-S1 and anti-RBD IgA levels
significantly increased before the changes in IgG and IgM levels. This
observation was consistent with the findings of a previous study, which
conducted a serological assay in a non-AA
cohort.\textsuperscript{13}Since spike proteins are integral to viral
entry into cells,\textsuperscript{14,15} IgA might play an important
role for effective control of the infection. The anti-S1 and anti-N IgG
levels subsequently increased, but they peaked at levels below the
cutoff values. The anti-RBD-specific IgG level was almost unchanged in
our patient. These differed from what was observed in other patients
with COVID-19 (mostly immunocompetent) in the University of Tokyo
Hospital, whose anti-SARS-CoV-2 IgG level increased significantly in all
cases.\textsuperscript{16} In addition, in a previous case series on
COVID-19 with AA, all four patients (with one on IST) had an elevated
anti-spike protein IgG level after COVID-19.\textsuperscript{3} However,
the effect of AA and IST on decreased IgG production and a favorable
outcome warrants additional investigation.

In summary, we reported a case of COVID-19 in a patient with AA
undergoing IST. Together with previous reports, our findings suggest
that AA patients do not necessarily have a higher risk of severe
COVID-19 compared with the general population. Future investigations are
needed to determine the optimal management for these patients and the
ability of patients with AA to produce sufficient protective antibodies.

\textbf{CONFLICT OF INTEREST STATEMENT}

The authors declare no conflict of interest.

\textbf{ETHICS STATEMENT}

The study protocol was approved by the ethics board of the University of
Tokyo (approval number: 2019300NI-3), and informed consent was obtained
from the legal guardians of the patient.

\textbf{ACKNOWLEDGEMENTS}

We would like to thank the COVID-19 team in the University of Tokyo
Hospital for their dedication to patient care and Editage
(www.editage.com) for English language editing.

\textbf{REFERENCES}

1. Dong Y, Mo X, Hu Y, et al. Epidemiology of COVID-19 Among Children in
China. \emph{Pediatrics.} 2020;145(6).

2. Akcabelen YM, Koca Yozgat A, Parlakay AN, Yarali N. COVID-19 in a
child with severe aplastic anemia. \emph{Pediatr Blood
Cancer.}2020;67(8):e28443.

3. Paton C, Mathews L, Groarke EM, et al. COVID-19 infection in patients
with severe aplastic anaemia. \emph{Br J Haematol.} 2021.

4. Keiffer G, French Z, Wilde L, Filicko-O'Hara J, Gergis U, Binder AF.
Case Report: Tocilizumab for the Treatment of SARS-CoV-2 Infection in a
Patient With Aplastic Anemia. \emph{Front Oncol.} 2020;10:562625.

5. Dixon L, Varley J, Gontsarova A, et al. COVID-19-related acute
necrotizing encephalopathy with brain stem involvement in a patient with
aplastic anemia. \emph{Neurol Neuroimmunol Neuroinflamm.} 2020;7(5).

6. Wang Y, Lu X, Chen T, Wang J. Lessons from a patient with severe
aplastic anemia complicated with COVID-19. \emph{Asian J
Surg.}2021;44(1):386-388.

7. Mazzini L, Martinuzzi D, Hyseni I, et al. Comparative analyses of
SARS-CoV-2 binding (IgG, IgM, IgA) and neutralizing antibodies from
human serum samples. \emph{J Immunol Methods.} 2021;489:112937.

8. Piccoli L, Park YJ, Tortorici MA, et al. Mapping Neutralizing and
Immunodominant Sites on the SARS-CoV-2 Spike Receptor-Binding Domain by
Structure-Guided High-Resolution Serology.
\emph{Cell.}2020;183(4):1024-1042.e1021.

9. Iyer AS, Jones FK, Nodoushani A, et al. Persistence and decay of
human antibody responses to the receptor binding domain of SARS-CoV-2
spike protein in COVID-19 patients. \emph{Sci Immunol.} 2020;5(52).

10. Minotti C, Tirelli F, Barbieri E, Giaquinto C, Donà D. How is
immunosuppressive status affecting children and adults in SARS-CoV-2
infection? A systematic review. \emph{J Infect.} 2020;81(1):e61-e66.

11. Filocamo G, Minoia F, Carbogno S, Costi S, Romano M, Cimaz R.
Absence of Severe Complications From SARS-CoV-2 Infection in Children
With Rheumatic Diseases Treated With Biologic Drugs. \emph{J
Rheumatol.}2020.

12. Mehta P, McAuley DF, Brown M, et al. COVID-19: consider cytokine
storm syndromes and immunosuppression.
\emph{Lancet.}2020;395(10229):1033-1034.

13. Ma H, Zeng W, He H, et al. Serum IgA, IgM, and IgG responses in
COVID-19. \emph{Cell Mol Immunol.} 2020;17(7):773-775.

14. Shang J, Wan Y, Luo C, et al. Cell entry mechanisms of
SARS-CoV-2.\emph{Proc Natl Acad Sci U S A.} 2020;117(21):11727-11734.

15. Yan R, Zhang Y, Li Y, Xia L, Guo Y, Zhou Q. Structural basis for the
recognition of SARS-CoV-2 by full-length human ACE2.
\emph{Science.}2020;367(6485):1444-1448.

16. Nakano Y, Kurano M, Morita Y, et al. Time course of the sensitivity
and specificity of anti-SARS-CoV-2 IgM and IgG antibodies for
symptomatic COVID-19 in Japan. \emph{Sci Rep.} 2021;11(1):2776.

\textbf{LEGENDS}

\textbf{FIGURE 1} \textbf{Clinical course of the patient}

Clinical course including treatment, absolute neutrophil and lymphocyte
counts, and SARS-CoV-2 serostatus are illustrated. Each bar and point
represent the period and timing in which drugs or blood transfusion were
administered.

ALC, absolute lymphocyte count; ANC, absolute neutrophil count; CS,
corticosteroids; CyA, cyclosporine; EPAG, eltrombopag; rATG, rabbit
antithymocyte globulin; RBC, red blood cell transfusion; Plt, platelet
transfusion; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2

\textbf{FIGURE 2} \textbf{Titers of anti-SARS-CoV-2 antibodies}

Each chart illustrates the transitions of IgG, IgM, and IgA antibodies
against each of S1, RBD, and N. The dashed lines depict the cutoff
values for each antibody determined as mentioned in the method section.

IgA, immunoglobulin A; IgG, immunoglobulin G, IgM immunoglobulin M, N,
nucleocapsid protein; RBD, receptor-binding domain; S1, S1 subunit of
spike protein; SARS-CoV-2, severe acute respiratory syndrome coronavirus
2\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/fig1/fig1}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/fig2/fig2}

\end{center}
\end{figure}

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

