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\begin{document}

\title{Proper assignation of reactivation in a COVID-19 recurrence initially
interpreted as a reinfection}



\author[1]{laura Pérez-Lago}%
\author[2]{helena Martínez}%
\author[2]{Jose Antonio pajares}%
\author[2]{Arantxa Diaz Gomez}%
\author[2]{Marina machado}%
\author[1]{Pedro Sola-Campoy}%
\author[3]{Marta Herranz}%
\author[2]{Maricela valerio}%
\author[2]{Maria Olmedo}%
\author[2]{Cristina Andres Zayas}%
\author[4]{Iñaki Comas}%
\author[5]{fernando Gonzalez Candelas}%
\author[2]{Rafael Bañares}%
\author[3]{Pilar Catalán}%
\author[3]{Patricia Muñoz}%
\author[2]{Darío García de Viedma}%
\affil[1]{Instituto de Investigación Sanitaria Gregorio Marañón}%
\affil[2]{Affiliation not available}%
\affil[3]{Hospital General Universitario Gregorio Marañón}%
\affil[4]{Instituto de Biomedicina de Valencia (IBV-CSIC)}%
\affil[5]{University of Valencia}%


\vspace{-1em}



  
  \date{May 26, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
A 77-year-old male (Case R) who had had a previous diagnosis of mild
COVID-19 episode, with fever without developing pneumonia or other
complications, was hospitalized 57 days later, due to an acute
cholangitis. He had a prolonged hospital stay with severe biliary and
infectious complications. On Day 23 post-admission, the patient
developed a second COVID-19 episode, now severe, with bilateral
pneumonia, multiorgan failure, and finally died. Initially, Case R
COVID-19 recurrence was interpreted as a reinfection due to the exposure
to a patient with whom he had shared the hospital room, who also had a
subsequent positive SARS-CoV-2 RT-PCR. However, whole genome sequencing
data indicated that both cases were infected by different strains and
clarified that case R recurrence corresponded to a reactivation of the
strain involved in his first episode. Case R reactivation had major
consequences, not only leading to a much more severe second episode, but
causing a subsequent transmission to another two hospitalized patients,
one of them with fatal resolution.%
\end{abstract}%



\sloppy


\textbf{Proper assignation of reactivation in a COVID-19 recurrence
initially interpreted as a reinfection}

Laura P\selectlanguage{ngerman}érez Lago\textsuperscript{1,2+}, Helena Martínez
Lozano\textsuperscript{2,3+}, Jose Antonio Pajares
Díaz\textsuperscript{2,3+}, Arantxa Díaz Gómez\textsuperscript{2,3+},
Marina Machado\textsuperscript{1,2}, Pedro J.
Sola-Campoy\textsuperscript{1,2}, Marta Herranz\textsuperscript{1,2,4},
Maricela Valerio\textsuperscript{1,2}, María
Olmedo\textsuperscript{1,2}, Cristina Andrés Zayas\textsuperscript{2,5},
Iñaki Comas\textsuperscript{6,7}, Fernando González
Candelas\textsuperscript{7,8}, Rafael Bañares\textsuperscript{9,10},
Pilar Catalán\textsuperscript{1,2,4}, Patricia
Muñoz\textsuperscript{1,2,4,9}, and Darío García de
Viedma\textsuperscript{1,2,4\textbf{*}} on behalf of Gregorio Marañón
Microbiology-ID COVID-19 Study Group

\textsuperscript{1}Servicio de Microbiología Clínica y Enfermedades
Infecciosas, Gregorio Marañón General University Hospital, Madrid, Spain

\textsuperscript{2}Instituto de Investigación Sanitaria Gregorio Marañón
(IiSGM), Madrid, Spain

\textsuperscript{3}Servicio de Digestivo, Gregorio Marañón General
University Hospital, Madrid, Spain

\textsuperscript{4}CIBER Enfermedades Respiratorias (CIBERES), Madrid,
Spain

\textsuperscript{5}Genomics Unit, Gregorio Marañón General University
Hospital, Madrid, Spain

\textsuperscript{6}Instituto de Biomedicina de Valencia-CSIC, Valencia,
Spain

\textsuperscript{7}CIBER Salud Pública (CIBERESP), Spain

\textsuperscript{8}Joint Research Unit ''Infection and Public Health''
FISABIO-University of Valencia, Institute for Integrative Systems
Biology (I2SysBio), Valencia, Spain

\textsuperscript{9}Departamento de Medicina, Universidad Complutense,
Madrid, Spain

\textsuperscript{10}CIBER Enfermedades hepáticas y digestivas
(CIBERehd). Madrid, Spain

\textbf{*Corresponding author:}\emph{dgviedma2@gmail.com}

Phone: (+34) 914265104

Postal address: C/Dr. Esquerdo 46 (28007), Madrid, Spain.

+: these authors contributed equally to this work

\textbf{Running title} : COVID-19 reactivation and nosocomial
transmission

\textbf{Summary}

A 77-year-old male (Case R) who had had a previous diagnosis of mild
COVID-19 episode, with fever without developing pneumonia or other
complications, was hospitalized 57 days later, due to an acute
cholangitis. He had a prolonged hospital stay with severe biliary and
infectious complications. On Day 23 post-admission, the patient
developed a second COVID-19 episode, now severe, with bilateral
pneumonia, multiorgan failure, and finally died. Initially, Case R
COVID-19 recurrence was interpreted as a reinfection due to the exposure
to a patient with whom he had shared the hospital room, who also had a
subsequent positive SARS-CoV-2 RT-PCR. However, whole genome sequencing
data indicated that both cases were infected by different strains and
clarified that case R recurrence corresponded to a reactivation of the
strain involved in his first episode. Case R reactivation had major
consequences, not only leading to a much more severe second episode, but
causing a subsequent transmission to another two hospitalized patients,
one of them with fatal resolution.

\textbf{Keyword} s: COVID-19, SARS-CoV-2, reactivation, nosocomial
transmission, WGS.

Word count Abstract: 163

Word count text: 1748

\textbf{Introduction}

Whole genome sequencing (WGS) has been essential to clarify a key aspect
in the COVID-19 pandemic, namely, the analysis of recurrences, allowing
to identify which are due to reinfections (Mulder et al., 2020; Tillett
et al., 2020). Genomic research has demonstrated the prolonged
persistence of viable SARS-CoV-2 in severely immunosuppressed patients
(Baang et al., 2020; Choi et al., 2020), but it has not equally been
used to support reactivations, and the scarce reports focus primarily on
clinical descriptions (Coppola, Annunziata, Carannante, Di Spirito, \&
Fiorentino, 2020). Furthermore, the potential relationship between
SARS-CoV-2 reactivation and associated nosocomial outbreaks has not been
described to date. In this study we present a SARS-CoV-2 reactivation
and its consequences in the nosocomial setting.

\textbf{Patients and Methods}

\textbf{Clinical data}

Baseline characteristics and clinical and laboratory parameters at
COVID-19 diagnosis and their outcome were obtained from their electronic
medical records. The study was approved by the ethical research
committee of Gregorio Marañón Hospital (REF: MICRO.HGUGM.2020-042)

\textbf{Diagnostic RT-PCRs}

Viral RNA was extracted and purified from 300 \selectlanguage{greek}μ\selectlanguage{english}L of nasopharyngeal
exudates with the aid of the KingFisher (Thermo Fisher Scientific,
Waltham, Massachusetts) instrument. Next, an RT-PCR was performed, using
the TaqPath COVID-19 CE-IVD RT-PCR kit (Thermo Fisher Scientific, USA).

\textbf{Whole genome sequencing}

Eleven \selectlanguage{greek}μ\selectlanguage{english}L of RNA were used as template for reverse transcription using
Invitrogen SuperScript IV reverse transcriptase (ThermoFisher
Scientific, Massachusetts, USA) and random hexamers (ThermoFisher
Scientific, Massachusetts, USA). Whole genome amplification of the
coronavirus was done with an Artic\_nCov-2019\_V3 panel of primers
(Integrated DNA Technologies, Inc., Coralville, Iowa, USA)
(artic.network/ncov-2019) and the Q5 Hot Start DNA polymerase (New
England Biolabs, Ipswich, Massachusetts, USA). Libraries were prepared
using the Nextera Flex DNA Library Preparation Kit (Illumina lnc,
California, USA) following manufacturer\selectlanguage{ngerman}´s instructions.

Libraries were quantified with the Quantus\selectlanguage{english} Fluorometer (Promega,
Wisconsin, USA), before being pooled at equimolar concentrations (4 nM).
Next, they were sequenced in pools of up to 17 libraries on the Miseq
system (Illumina Inc, California, USA) and the MiSeq Reagent Micro kit
v2 (2x151pb) or in pools of up to 96 libraries with the MiSeq Reagent
(2x201 pb).

FastQ files above the GISAID thresholds were deposited at GISAID
EPI\_ISL\_654287, EPI\_ISL\_654203, EPI\_ISL\_654284, EPI\_ISL\_654176
and EPI\_ISL\_1173765. An in-house analysis pipeline was applied to
analyse the sequencing reads. The pipeline can be accessed at
https://github.com/pedroscampoy/covid\_multianalysis. Briefly, the
pipeline goes through the following steps: 1) removal of human reads
with Kraken
{[}https://genomebiology.biomedcentral.com/articles/10.1186/gb-2014-15-3-r46{]};
2) pre-processing and quality assessment of fastq files using fastp
{[}https://academic.oup.com/bioinformatics/article/34/17/i884/5093234{]}
v0.20.1 (arguments: --cut tail, --cut-window-size, --cut-mean-quality ,
-max\_len1 ,-max\_len2 ) and fastQC v0.11.9 {[}Andrews S.; S Bittencourt
a, ``FastQC: a quality control tool for high throughput sequence data --
ScienceOpen,'' Babraham Inst., p.
http://www.bioinformatics.babraham.ac.uk/projects/, 2010.{]}; 3) mapping
with bwa v0.7.17 {[}H. Li and R. Durbin, ``Fast and accurate short read
alignment with Burrows-Wheeler transform,'' Bioinformatics, vol. 25, no.
14, pp. 1754--1760, 2009.{]} and variant calling using IVAR v1.2.3
{[}https://genomebiology.biomedcentral.com/articles/10.1186/s13059-018-1618-7{]}
using Wuhan-1 sequence (NC\_045512.2) as reference; 4) Recalibration of
punctual low coverage positions using joint variant calling. When
necessary, informative non-covered positions were analysed by standard
Sanger sequencing with the corresponding flanking primers from the ARTIC
set.

\textbf{Results}

Our case (Case R, Figure 1) was a 77-year-old male with hypertension and
dyslipidaemia, a diagnosis of cutaneous B-cell lymphoma in remission, a
previous stroke, and chronic obstructive pulmonary disease associated to
mild interstitial lung disease without exacerbation nor need of
supplemental oxygen. His first positive SARS-CoV-2 RT-PCR was on July
28, 2020 when he had a mild infection with fever without developing
pneumonia or other complications. Hospital admission was not required.
SARS-CoV-2 serology was not performed at that time.

On September 1, he was admitted to the hospital due to an acute
obstructive cholangitis secondary to choledocholithiasis that was
removed by endoscopy. The patient received piperacillin-tazobactam.
After the endoscopic procedure, he developed mild acute pancreatitis,
hemobilia, and acute kidney injury related to acute tubular necrosis. In
addition, he developed catheter-related \emph{Enterococcus
faecium}bacteraemia successfully treated with vancomycin. During this
time, he obtained two negative SARS-CoV-2 RT-PCR tests (September 1 and
14, Figure 1).

On Day 23 following admission, extensive bilateral lung opacities were
identified in a control abdominal computed tomography (CT). After these
unexpected radiological findings, SARS-CoV-2 RT-PCRs were performed for
two consecutive days, both positive (Ct 19, Ct 21). IgG SARS CoV-2
serology was negative (Figure 1).

Case R developed mild dyspnoea and hypoxemia (oxygen saturation of 92\%
at room air). He received remdesivir for five days and dexamethasone 20
mg once daily for four days. After a slight improvement, on Day 29, he
developed fever and respiratory worsening. On Day 31, high-flow oxygen
therapy and a single 400 mg dose of tocilizumab (IL-6 level: 226pg/mL)
were administered. The patient was transferred to the ICU where he
received full ventilatory support and continuous changing between prone
and supine positions. However, the patient rapidly developed multiorgan
failure with hemodynamic instability, mixed metabolic and respiratory
acidosis, and renal impairment requiring continuous renal replacement
therapy. Body CT scan revealed non-specific colitis and worsening of the
bilateral pulmonary opacities with pleural effusion. A colonoscopy ruled
out ischemic colitis. Despite all therapeutic interventions, the patient
developed refractory multi-system organ failure and finally died on Day
34. Retrospectively, we recovered three sera specimens (from days 23,
the day the nasopharyngeal RT-PCR result was positive, 27, and 30) and
all were positive for SARS-CoV-2 RT-PCR. Clinical outcomes are shown in
Figure 1.

\textbf{Whole genome sequencing analysis}

Prior to having the WGS data, several findings, i.e., chronology of
SARS-CoV-2 infections, dates of symptom onset, positive SARS-CoV-2
RT-PCRs, and room coincidences, led clinicians to assume that Case R
recurrence was a reinfection due to the exposure to a patient with whom
he had shared the hospital room (Case A) and who had been admitted 11
days before due to an intestinal obstruction, had a bilateral pneumonia
and subsequent positive SARS-CoV-2 RT-PCR. However WGS data (obtained in
a larger study analysing a wide nosocomial outbreak in the
Gastroenterology ward, under evaluation) indicated that fully different
strains were identified in Case A and Case R (Figure 2). In addition,
Case R was part of Cluster which also included Cases S and T, infected
by an identical strain (0 SNPs, Figure 2). Cases S and T had shared a
room, but Case R at the time of his positive-RT-PCR was in a different
one. However, tracking back his previous movements revealed that Case R
had shared room with case S seven days before, confirming a link between
them; SARS-CoV-2 infection in Case S had a fatal outcome.

WGS data ruled out our initial hypothesis of reinfection after
nosocomial exposure and led us to consider, alternatively, Case R as a
reactivation, causing a subsequent nosocomial transmission. The
sequences of the positive specimens collected from Case R first and
second episodes (July and September, 2020) belonged to the same lineage
(B.1.177) and showed nearly identical sequences; they shared 16 SNPs and
differed in two (Figure 3, Supplementary Table). Given the marked
diversity of circulating SARS-CoV-2 in the second COVID-19 wave, the
high similarity between the sequences strongly supports that Case R
recurrence most likely corresponded to a reactivation.

\textbf{Discussion}

This study shows the importance of WGS-based analysis to correctly
understand COVID-19 recurrences and, additionally, the true links within
nosocomial transmission events. This technique provided key data to
describe a COVID-19 reactivation, which was subsequently responsible for
another two nosocomial cases.

The similarities between the strains infecting Case R in the July and
September episodes may be explained by either a persistent infection or
a reactivation. Persistence was ruled out because the patient fully
recovered from mild clinical symptoms experienced during his first
episode. Furthermore, X-rays at admission did not show abnormal
SARS-CoV-2-related findings and two sequential negative PCRs just before
being diagnosed again in September (at admission and 14 days later) were
obtained. Finally, during the 23 days of hospital stay before
reactivation, the patient had close contact with four roommates, none of
which had a COVID-19 diagnosis. All these findings rule out a hypothesis
of persistence.

An alternative explanation for the high sequence similarities between
the specimens collected during the two episodes experienced by Case R
would be reactivation. The subtle differences (two different SNPs and 16
identical SNPs) found for this case are similar to those described in a
reactivation reported elsewhere (Yadav et al., 2021). Reservoirs for
SARS-CoV-2 after the resolution of a COVID-19 episode have not been
defined yet. The involvement of extra-pulmonary tissues (eyes,
gastrointestinal tract, liver, and brain) has been reported (Meinhardt
et al., 2020; Paizis et al., 2005; Wang et al., 2020), due to the
ubiquity of the ACE2 receptors. Moreover, SARS-CoV-2 RNA has been
detected in anal swabs for 42 days in an asymptomatic carrier, while
nasopharyngeal swabs were negative (Jiang et al., 2020). The presence of
SARS-CoV-2 in non-respiratory tissues suggests that further studies are
needed to identify other viral reservoirs (Kalkeri, Goebel, \& Sharma,
2020).

If the reservoir hypothesis were correct, we would expect reactivations
to be mainly associated to immunosuppression, which would trigger the
replication of the latent strain. Few studies have proposed reactivation
as the explanation for COVID-19 recurrence (Coppola et al., 2020;
Lancman, Mascarenhas, \& Bar-Natan, 2020), some involving
immunosuppression. However, only two were supported with viral genome
analyses (Molina et al., 2020) (Yadav et al., 2021). Several factors
suggest the presence of immunosuppression in Case R. Firstly, he had
stayed hospitalized 23 days suffering of severe conditions before his
first positive RT-PCR. Acute care settings is a risk factor of
malnutrition. Before the diagnosis of COVID-19, Case R had lymphopenia
for 12 days; this may impair immunity, a factor associated to increased
morbidity and mortality (Liu Y, 2020; Ziadi A, 2020). Secondly, the
patient suffered of severe gastrointestinal conditions (acute
cholangitis, post-ERCP acute pancreatitis, and gastrointestinal bleeding
requiring blood transfusion) that could have worsened his immune system.
Finally, he presented two infections (cholangitis and a catheter-related
infection) and acute kidney injury that might have further worsened his
already weakened immune system.

A relevant retrospective finding in Case R is the positive SARS-CoV-2
RT-PCR in three sera specimens taken the same day he had his first
diagnostic SARS-CoV-2 RT-PCR, and four and six days later. SARS-CoV-2
may be detected in plasma samples from patients with respiratory disease
and this may have value to predict the severity of the disease (Veyer et
al., 2020). However, this has not been found close to diagnosis, even in
cases with pneumonia (Nijhuis et al., 2020). Therefore, the presence of
SARS-CoV-2 in plasma in the second episode experienced by Case R, would
suggest that we are not facing a new infection but a likely longer-term
disease, which may support the reactivation scenario .

In summary, we report genomic viral analysis allowed to identify a
reactivation case with major consequences, leading to a more severe
second episode with fatal resolution and subsequent nosocomial
transmission of the same strain with an additional COVID-19-related
death.

\textbf{Acknowledgements}

This work was supported by \emph{Instituto de Salud Carlos III} (Ref
COV20/00140: SeqCOVID - \emph{Consorcio para la epidemiologia genomica
de SARS-CoV-2 en Espana} ) and by \emph{Consejo Superior de
Investigaciones Cientificas} (\emph{CSIC} ) (\emph{PTI Salud Global} ).
LPL holds a Miguel Servet Contract CP15/00075). We are grateful to
Dainora Jaloveckas (cienciatraducida.com) for editing and proofreading
assistance.

\textbf{Conflict of interests}

The authors do not have commercial or other associations that might pose
a conflict of interest.

\textbf{Data availability}

The data that support the findings of this study (FastQ files) are
openly available in GISAID at https://www.gisaid.org/ . Reference
numbers EPI\_ISL\_654287, EPI\_ISL\_654203, EPI\_ISL\_654284,
EPI\_ISL\_654176 and EPI\_ISL\_1173765.

\textbf{Figures}

\textbf{Figure 1.} Clinical timeline for Case R. ERCP: endoscopic
retrograde cholangiopancreatography; RT-PCR: Reverse-transcription
polymerase chain reaction; S: serum sample; NP nasopharyngeal sample;
(+) Positive result; (-) Negative result; RBC: red blood cells
transfusion. CT: computerized axial tomography~scan. MO failure:
multiorgan failure; HFNC: high-flow nasal cannulas; O. intubation:
orotracheal intubation

\textbf{Figure 2.} Network of relationships obtained from whole genome
sequencing analysis for the outbreak strains. Each dot corresponds to a
single nucleotide polymorphism. When two or more cases share identical
genome (zero single nucleotide polymorphisms between them) they are
included in the same box. mv: median vector: not sampled recent common
ancestor for the two branches. ANC: Wuhan-1 reference strain.

\textbf{Figure 3.} Distribution along the SARS-CoV-2 chromosome of the
single nucleotide polymorphisms identified in the two sequential
episodes of Case R. Each vertical black bar corresponds to a single
nucleotide polymorphism.

\textbf{Gregorio Maranon Microbiology-ID COVID-19 Study Group}

Adan-Jimenez (Javier), Alcala (Luis), Aldamiz (Teresa), Alonso
(Roberto), Alvarez (Beatriz), Alvarez-Uria (Ana), Arias (Alexi), Arroyo
(Luis Antonio), Berenguer (Juan), Bermudez (Elena), Bouza (Emilio),
Burillo (Almudena), Candela (Ana), Carrillo (Raquel), Catalan (Pilar),
Cercenado (Emilia), Cobos (Alejandro), Diez (Cristina), Escribano
(Pilar), Estevez (Agustin), Fanciulli (Chiara), Galar (Alicia), Garcia
(Ma Dolores), Garcia de Viedma (Dario), Gijon (Paloma), Gonzalez
(Adolfo), Guillen (Helmuth) Guinea (Jesus), Haces (Laura Vanessa),
Herranz (Marta), Kestler (Martha), Lopez (Juan Carlos), Losada (Carmen
Narcisa), Machado (Marina), Marin (Mercedes), Martin (Pablo), Montilla
(Pedro), Munoz (Patricia), Olmedo (Maria), Padilla (Belen), Palomo
(Maria), Parras (Francisco), Perez-Granda (Maria Jesus), Perez-Lago
(Laura), Perez (Leire), Pescador (Paula), R Maus (Sandra), Reigadas
(Elena), Rico-Luna (Carla Margarita), Rincon (Cristina), Rodriguez
(Belen), Rodriguez (Sara), Rodriguez-Grande (Cristina), Rojas (Adriana),
Ruiz-Serrano (Maria Jesus), Sanchez (Carlos), Sanchez (Mar), Serrano
(Julia), Sola Campoy (Pedro J), Tejerina (Francisco), Valerio
(Maricela), Veintimilla (Ma Cristina), Vesperinas (Lara), Vicente
(Teresa), de la Villa (Sofia).

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