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\begin{document}

\title{HUMAN ASTROVIRUS VA1 ENCEPHALITIS IN PEDIATRIC CANCER PATIENTS: REPORT
OF TWO CASES AND REVIEW OF THE LITERATURE}



\author[1]{Sakshi Bami}%
\author[1]{Jennifer Hidinger}%
\author[1]{Arshia Madni }%
\author[1]{Virginia Hargest}%
\author[1]{Stacey Schultz-cherry}%
\author[1]{Valerie Cortez}%
\author[2]{Shane Cross}%
\author[2]{Deborah Ward}%
\author[1]{Randall Hayden}%
\author[2]{Jeffrey Rubnitz}%
\author[2]{Ching-Hon Pui}%
\author[2]{Raja Khan}%
\author[2]{Diego Hijano}%
\affil[1]{St Jude Children's Research Hospital}%
\affil[2]{St. Jude Children's Research Hospital}%


\vspace{-1em}



  
  \date{May 28, 2024}


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\selectlanguage{english}
\begin{abstract}
Novel human astrovirus (HAstV) strains have been recently shown to cause
fatal encephalitis in immunocompromised patients. We report two cases
from our institution. A 2-year old female undergoing treatment for acute
lymphoblastic leukemia and a 9-year old male with refractory acute
myeloid leukemia. Both were found to have HAstV-VA1 in the cerebrospinal
fluid (CSF) by metagenomics next generation sequencing (m-NGS) after the
initial evaluation did not reveal any etiology. Patient 1 remains alive,
in remission, showing gradual neurological improvement after treatment
with steroids, intravenous immunoglobulin, ribavirin, nitazoxanide and
Peg-INF-alpha 2b. Patient 2 died 7 weeks later while receiving
palliative care.%
\end{abstract}%



\sloppy


\textbf{INTRODUCTION}

Encephalitis is characterized by brain dysfunction due to inflammation
of the brain parenchyma and is associated with significant morbidity and
mortality. Despite significant advances in diagnostic methods, the
etiology remains unknown in about 60\% of cases.\textsuperscript{1-3}

Immunocompromised patients with encephalitis pose unique diagnostic and
therapeutic challenges. They are more likely to have atypical
presentations from common pathogens and are at higher risk of infections
by novel agents.\textsuperscript{4}

Classical human astroviruses (HAstV) are gastrointestinal pathogens
responsible for approximately 10\% cases of acute, non-bacterial
gastroenteritis in children.\textsuperscript{5-7} Recently discovered,
divergent strains of HAstV, MLB and VA1, also cause self-limited
diarrheal illnesses.\textsuperscript{6,8} However, HAstVs have also been
reported to cause fatal encephalitis, primarily in immunocompromised
hosts. To date, 10 cases of HAstV central nervous system (CNS) infection
have been reported.\textsuperscript{9-17} The most frequently identified
strain has been VA1.\textsuperscript{9,11-14} We report 2 cases of
HAsV-VA1 encephalitis in children with cancer at our institution and
perform a literature review of previously published cases.

\textbf{METHODS}

Demographic and clinical information was abstracted. We performed ELISAs
to identify and quantify HAstV-1 and HAstV-VA1 specific antibodies in 9
polyvalent IVIG lots. In addition, we performed neutralization as
described elsewhere. \textsuperscript{18} (Supplemental methods) The
study was approved by St Jude's Institutional Review Board. Consent from
parents of Patient 2 was waived and verbal consent from parents of
Patient 1 was obtained.

\textbf{CASE REPORT}

\textbf{PATIENT 1}

A 2-year-old female with standard risk B-cell acute lymphoblastic
leukemia (B-ALL) and CNS leukemia at diagnosis developed myoclonic
twitching during week 22 of continuation chemotherapy. She became
increasingly somnolent, irritable, and developed bouts of agitation,
difficulty in walking, inability to communicate, and dysphagia. A
magnetic resonance imaging (MRI) of the brain demonstrated cerebral
volume loss and a nonspecific ill-defined FLAIR hyperintensity in the
cerebral white matter. Electroencephalogram (EEG) showed diffuse
slowing. CSF bacterial and fungal cultures were negative (Supplemental
Table 1). PCR panel did not detect micro-organisms, autoimmune panel
resulted negative, and cytology showed no evidence of leukemia. For
presumed autoimmune encephalitis, she was treated with intravenous
immunoglobulin (IVIG) with no improvement. CSF m-NGS resulted positive
for HAstV-VA1. Management was based on a previously published
case\textsuperscript{11} and included ribavirin, methylprednisolone,
IVIG and Peg-INF-alpha 2b. In addition, we added oral nitazoxanide due
to its efficacy in vitro against HAstV-VA1.\textsuperscript{19,20} Due
to side effects, Peg-INF-alpha 2b was discontinued after 3 doses.
(Supplemental Fig 1)

To further optimize her treatment, we tested 9 lots of IVIG for HAstV
antibodies and did not find any significant difference (Supplemental
Fig.2A). None showed significant viral neutralization (Supplemental
Fig.2B, 2C).

MRI demonstrated T2 prolongation in thalami and restricted water
diffusion consistent with acute necrotizing encephalitis that improved
over time. (Fig.1) She was discharged after 108 days of hospitalization
on PO ribavirin, nitazoxanide and IVIG. Her chemotherapy was held and
restarted 15 days after astrovirus treatment initiation. Dexamethasone
and vincristine were omitted from therapy to prevent further CNS damage.
She was treated for astrovirus infection for approximately 8 months
(Supplemental Fig.1) and has shown gradual yet significant neurologic
improvement. At her most recent follow up, 16 months after diagnosis,
she could socially interact but was minimally verbal, remained without
seizures with improving hypertonia, and demonstrated clinical
improvement even after stopping astrovirus directed therapy.

\textbf{PATIENT 2}

A 9-year-old male with KMT2A-rearranged relapsed refractory acute
myeloid leukemia developed respiratory syncytial virus (RSV) infection
and was admitted for neutropenic fever.

Shortly after admission he developed altered mental status, including
periods of agitation and confusion alternating with being overly
sedated. Head CT showed cerebral volume loss. EEG showed diffuse slowing
consistent with encephalopathy. CSF evaluation showed negative bacterial
and fungal cultures. PCR panel was negative for micro-organisms, and
cytology showed no leukemia involvement (Supplemental Table 1). He was
treated with broad spectrum antibiotics for neutropenic fever, acyclovir
for presumed herpes simplex virus encephalitis and PO ribavirin for RSV
infection. He received IVIG (1g/kg/dose x 2 days) for presumed
autoimmune encephalitis with no benefit. CSF m-NGS was positive for
HAstV-VA1.

Because of his incurable leukemia, he remained inpatient for palliative
care. Treatment for astrovirus infection was aligned with his goals of
care with IVIG (1 g/kg/dose thrice weekly for one week then weekly
thereafter) and oral nitazoxanide (250 mg orally twice daily). He had
progressive neurologic deterioration, developed renal and respiratory
failure and died 7 weeks after diagnosis. Family refused autopsy.

\textbf{DISCUSSION}

Divergent strains of astroviruses are known to cause infection in
humans, including classical HAstV (types 1-8), MLB and VA1
viruses.\textsuperscript{5} Studies show 90\% and 65\% seropositivity
against classical HAstV and VA1 and close to 100\% seropositivity in
adulthood for MLB1 suggesting that viral exposure is common and frequent
throughout life.\textsuperscript{21-24} These pathogens have recently
been found to cause CNS infection mostly in immunocompromised hosts,
especially VA1. Astrovirus encephalitis was first described in 2010.
Since then, a total of 10 cases of astrovirus CNS infection have been
reported (Table 1).\textsuperscript{9-17}

All five patients with HAstV-VA1 infection were
immunocompromised\textsuperscript{9,11-14} and were diagnosed with
m-NGS; in only 2 cases the virus was identified before the patient
died.\textsuperscript{11,13}One patient was treated with ribavirin,
steroids, IVIG and Peg-INF survived but recovered with cognitive and
movement sequelae.\textsuperscript{11} Two of three cases with MLB
encephalitis recovered fully, one was an immunocompetent
host.\textsuperscript{10,15}One patient with classical strain (HAstV-4)
died while the other was an immunocompetent host and
recovered.\textsuperscript{16,17} Hence, there seems to be variability,
not only on the host characteristics but also on the virulence among
strains.

We report two children who developed encephalitis with HAstV VA1 strain
while undergoing treatment for leukemia. In both patients, traditional
diagnostic tests were negative, and the virus was identified using m-NGS
on the CSF. This unbiased technique, not limited by specific targets,
enables the identification of novel
pathogens.\textsuperscript{25}Despite its potential, m-NGS is not
routinely utilized. Considerations of costs, reimbursement, turnaround
time, and regulations remain major hurdles.\textsuperscript{25}
Interpretation of m-NGS can be difficult and discerning between a
contaminant, colonizer or pathogen can be
challenging.\textsuperscript{25}

Patient 1 was managed based on the treatment outlined by Fr\selectlanguage{ngerman}émond et
al\textsuperscript{11} which resulted in the only surviving case of VA1
encephalitis to date. Our patient was treated for about 8 months and
survived. She remains with significant neuropsychiatric sequelae and
dependent for activities of daily living. The extent to which she will
recover neurologically remains unknown. Given the paucity of information
in treating this severe infection, we attempted to optimize her
treatment by including nitazoxanide, based on in vitro
data\textsuperscript{19,20} and analyzing commercially available IVIG.
While we observed VA1 and HAstV-1 antibodies in all IVIG lots, there was
no significant differences in neutralizing activity. These results show
that commercially available IVIG lots have HAstV antibodies but their
role in this setting remains to be determined.

HAstV should be considered in the differential of acute encephalitis in
immunosuppressed patients. m-NGS is not routinely accessible but can be
vital in yielding a definitive diagnosis when conventional tests are
inconclusive. We emphasize the importance of early diagnosis due to high
morbidity and mortality associated with HAstV encephalitis, with
potential improved outcomes with the therapy described in surviving
patients. The true extent of the pathogenesis of HAstV among
immunocompromised patients is unclear and needs to be elucidated with
further studies.

\textbf{CONFLICT OF INTEREST}

Shane J cross is a consultant for Lexicomp. Randall T Hayden serves on
the advisory board for Roche molecular, Quidel and Inflammatix. The
other authors have no relevant conflict of interest.

\textbf{ACKNOWLEDGEMENTS}

None

\textbf{REFERENCES}

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with X-linked agammaglobulinemia. \emph{Emerg Infect
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16. Koukou G, Niendorf S, Hornei B, Schlump JU, Jenke AC, Jacobsen S.
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18. Janowski AB, Wang D. Infection and Propagation of Astrovirus VA1 in
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20. Hargest V, Sharp B, Livingston B, Cortez V, Schultz-Cherry S.
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21. Burbelo PD, Ching KH, Esper F, et al. Serological studies confirm
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23. Koopmans MP, Bijen MH, Monroe SS, Vinjé J. Age-stratified
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24. Kriston S, Willcocks MM, Carter MJ, Cubitt WD. Seroprevalence of
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\textbf{LEGENDS}

Figure 1

MRI images at the beginning of illness (A1, B1, C1), end of
2\textsuperscript{nd} week of illness (A2, B2, C2) and 8-weeks after
onset of illness (A3, B3, C3). A1-3 are T1 weighted images showing no
abnormality except increased generalized atrophy 2-weeks into the
illness (A2) and some recovery by 8-weeks (A3). Diffusion weighted
imaging (ADC map) showing restricted diffusion in bilateral thalami
(arrows) 2-weeks into the illness (B2) and no diffusion restriction
early in course (B1) and at recovery (B3). FLAIR imaging showing a hint
of increased signal in thalami early in the disease (C1), more
pronounced increased signal 2-weeks into the illness (C2), and recovery
by 8-weeks (C3). Note increased generalized brain atrophy 2-weeks into
the illness (A2, B2, C2) with some recovery by 8-weeks (A3, B3, C3).

Table 1

Cases of astrovirus central nervous system infection reported in the
literature\selectlanguage{english}
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