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\begin{document}

\title{SARS-CoV-2 re-positivity within the first 3 months of COVID-19 recovery;
probable re-infection}



\author[1]{sara sadr}%
\author[2]{Melika Arab Bafrani}%
\author[2]{Alireza Abdollahi}%
\author[2]{SeyedAhmad SeyedAlinaghi}%
\author[2]{Esmaeil Mohammadnejad}%
\author[2]{Roghieh Hossienzade}%
\author[2]{Fereshteh Shahmari Golestan}%
\author[2]{Zahra Ahmadinejad}%
\author[3]{Mohammadreza  Salehi}%
\author[2]{Mohammad Javaherian}%
\author[2]{Elahe kimyaee}%
\author[4]{Fatemeh Jafari}%
\author[2]{Fereshteh Ghiasvand}%
\affil[1]{Mazandaran University of Medical Sciences}%
\affil[2]{Tehran University of Medical Sciences}%
\affil[3]{Imam Khomeini Hospital Complex}%
\affil[4]{Iran University of Medical Sciences}%


\vspace{-1em}



  
  \date{June 07, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
Objectives Possibility of reinfection with SARS-CoV-2 changes our view
on herd immunity and vaccination, and can impact worldwide quarantine
policies. We performed RT-PCR follow-up studies on recovered patients to
assess possible development of reinfections and re-positivity. Method
During a 6-month period, 202 PCR-confirmed recovering COVID-19 patients
entered this study. Follow-up RT-PCR tests and symptoms assessment were
performed one month after the initial Positive results. patients who
tested negative were tested again one and three months later. The Serum
IgG and IgM levels were measured in the last follow-up session. Results
In the first two follow-up sessions, 82 patients continued their
participation, of which four patients tasted positive. In the second
follow-up 44 patients participated, three of whom tested positive. None
of the patients who tested positive in the first and second follow-up
session were symptomatic. In the last session, 32 patients were tested
and four patients were positive, three of them were mildly symptomatic
and all of them were positive for IgG. Conclusion A positive RT-PCR in a
recovering patient may represent reinfection. While we did not have the
resources to prove reinfection by genetic sequencing of the infective
viruses, we believe presence of mild symptoms in the three patients who
tested positive over 100 days after becoming asymptomatic, can be
diagnosed as reinfection. The IgG may have abated the symptoms of the
reinfection, without providing complete protection.%
\end{abstract}%



\sloppy


\textbf{SARS-CoV-2 re-positivity within the first 3 months of COVID-19
recovery; probable re-infection}

\textbf{Running title:}

SARS-CoV-2 re-positivity, 3-months post-remission

\textbf{Author names and affiliations:}

Sara Sadr\textsuperscript{1}, Melika Arab Bafrani\textsuperscript{2},
Alireza Abdollahi\textsuperscript{3}, SeyedAhmad
SeyedAlinaghi\textsuperscript{4}, Esmaeil
Mohammadnejad\textsuperscript{5}, Roghieh
Hossienzade\textsuperscript{6}, Fereshteh Shahmari
Golestan\textsuperscript{7}, Zahra Ahmadinejad\textsuperscript{8},
Mohamadreza Salehi\textsuperscript{9}, Mohammad
Javaherian\textsuperscript{10}, Elahe Kimyaee\textsuperscript{11},
Fatemeh Jafari\textsuperscript{12}, Fereshteh
Ghiasvand\textsuperscript{8*}

\begin{enumerate}
\tightlist
\item
  School of medicine, Mazandaran University of Medical Sciences, Sari,
  Iran.
\item
  Students\textsuperscript{'} scientific Research Center (SSRC), school
  of medicine, Tehran University of Medical Sciences, Tehran, Iran.
\item
  Department of Pathology, school of medicine, Imam Khomeini Hospital
  Complex, Tehran University of Medical Sciences, Tehran, Iran.
\item
  Iranian Research Center for HIV/AIDS, Iranian Institute for Reduction
  of High Risk Behaviors, Tehran University of Medical Sciences,
  Tehran, Iran
\item
  Assistant Professor of Nursing Education, Department of
  Medical-Surgical Nursing and Basic Science, School of Nursing and
  Midwifery, Tehran University of Medical Sciences, Tehran, Iran.
\item
  MSc, Student in nursing management leader ship, Imam Khomeini
  Hospital Complex, Tehran University of Medical Sciences, Tehran,
  Iran.
\item
  Nurse Working in Department of Infectious Diseases, Tehran University
  of Medical Sciences, Tehran, Iran.
\item
  Liver Transplantation Research Center, Department of Infectious
  Diseases, Imam Khomeini Hospital Complex, Tehran University of
  Medical Sciences, Tehran, Iran.
\item
  Department of Infectious Diseases, Imam Khomeini Hospital Complex,
  Tehran University of Medical Sciences, Tehran, Iran.
\item
  Department of Physiotherapy, School of Rehabilitation, Liver
  Transplantation Research Center, Imam Khomeini Hospital Complex,
  Tehran University of Medical Sciences, Tehran, Iran.
\item
  Central Laboratory, Imam Khomeini Hospital Complex, Tehran University
  of Medical Sciences, Tehran, Iran.
\item
  School of medicine, Iran University of Medical Sciences, Tehran, Iran.
\end{enumerate}

\textbf{*Corresponding author:}

Fereshteh Ghiasvand, M.D.

Liver Transplantation Research Center, Department of Infectious
Diseases, Imam Khomeini Hospital Complex, Keshavarz boulevard, Tehran
University of Medical Sciences, Tehran, Iran.

Tel: +98 21 61192659,

Fax: +98 21 66581657,

E-mail: ghiasvand\_62@yahoo.com

\textbf{Authors' emails:}

Sara Sadr: sadrsara.md@gmail.com Melika Arab Bafrani:
melika.arab92@gmail.com Alireza Abdollahi: alireza.a1345@yahoo.com
SeyedAhmad SeyedAlinaghi: s.a.alinaghi@gmail.com Esmaeil Mohammadnejad:
asreno1358@yahoo.com Roghieh Hossienzade: hroghieh@gmail.com Fereshteh
Shahmari Golestan: fshahmari441@gmail.com Zahra Ahmadinejad:
ahmadiz@tums.ac.ir Mohamadreza Salehi: salehi.mohamad3@gmail.com
Mohammad Javaherian: javaherian\_m@razi.tums.ac.ir Elahe kimyaee:
e.kimyaee1991@gmail.com Fatemeh Jafari: fatemejafari72@gmail.com
Fereshteh Ghiasvand: ghiasvand\_62@yahoo.com

\textbf{Acknowledgements}

This research has been financially supported by Tehran University of
Medical Sciences \& health services grant {[}grant number:
99-1-101-47279{]}. The supporting institution had no involvement in
study design; in the collection, analysis and interpretation of data; in
the writing of the report; and in the decision to submit the article for
publication. Authors also would like to appreciate the support and
constructive comments of the methodologist(s) research development
office, Imam khomeini Hospital complex. We would like to thank our
patients for their time and cooperation, and also the medical staff of
the Imam Khomeini Hospital Complex who helped facilitated the process of
our research.

\textbf{Conflict of Interest:} none

\textbf{Abstract:}

Objectives

Possibility of reinfection with SARS-CoV-2 changes our view on herd
immunity and vaccination, and can impact worldwide quarantine policies.
We performed RT-PCR follow-up studies on recovered patients to assess
possible development of reinfections and re-positivity.

Method

During a 6-month period, 202 PCR-confirmed recovering COVID-19 patients
entered this study. Follow-up RT-PCR tests and symptoms assessment were
performed one month after the initial Positive results. patients who
tested negative were tested again one and three months later. The Serum
IgG and IgM levels were measured in the last follow-up session.

Results

In the first two follow-up sessions, 82 patients continued their
participation, of which four patients tasted positive. In the second
follow-up 44 patients participated, three of whom tested positive. None
of the patients who tested positive in the first and second follow-up
session were symptomatic. In the last session, 32 patients were tested
and four patients were positive, three of them were mildly symptomatic
and all of them were positive for IgG.

Conclusion

A positive RT-PCR in a recovering patient may represent reinfection.
While we did not have the resources to prove reinfection by genetic
sequencing of the infective viruses, we believe presence of mild
symptoms in the three patients who tested positive over 100 days after
becoming asymptomatic, can be diagnosed as reinfection. The IgG may have
abated the symptoms of the reinfection, without providing complete
protection.

\textbf{Keywords:}

COVID-19, Reinfection, Virus Shedding, Polymerase Chain Reaction,
Seroconversion

\textbf{Introduction}

Since November 2019, the Severe Acute Respiratory Syndrome Coronavirus 2
(SARS-CoV-2) has infected more than 134 million people worldwide and has
caused more than 2 million deaths {[}1{]}. No curative drug or specific
treatment is known to have considerable efficacy against this infection;
but, currently over 200 candidate vaccines have been acknowledged by the
WHO {[}2-4{]}.

Our experience with other respiratory infections, such as those caused
by influenza and seasonal respiratory infections caused by coronavirus
lead us to believe that the natural immunity after an episode of
infection with this virus may not cause long-time immunity {[}5{]}. Some
studies have shown a substantial wane in antibody levels within a few
months after remission {[}6, 7{]}; and the studies that report
re-infection further question the efficacy and longevity of the acquired
immunity after infection with this virus. Several cases of suspected and
proven instances of re-infection with SARS-CoV-2 have been reported in
immunocompetent patients in different age groups, in patients with
different levels of antibody response {[}8-10{]}.

The duration of sustained antibody response after an episode of
infection is generally a representative of the immune protection that
can be achieved by vaccination against the same virus {[}11{]}. Waning
of the IgG antibodies that are naturally produced in a patient after
infection with SARS-CoV-2, undermines the supposed efficacy of
vaccination; especially since cases of symptomatic re-infection with
replication-competent virus have been reported within the first 6 months
after the initial infection {[}12, 13{]}.

To this date, thousands of distinct variants of SARS-CoV-2 have been
identified with over 400 variants in the spike protein, which is
presumably the binding cite of neutralizing antibodies {[}14{]}.
Besides, in many cases of re-infection, genetic sequencing has revealed
a different clade of the virus to be the causative pathogen {[}15-18{]}.
We can assume---as is the case with infections caused by rhinoviruses
and influenza---that the protective activity of antibodies is limited to
each specific subtype of the virus, this can possibly explain why
re-infection can occur in presence of detectable levels of IgG {[}19,
20{]}.

In this study we investigated the rate of symptomatic and asymptomatic
re-positivity with SARS-CoV-2 in recovering patients for up to four
months after the initial diagnosis of Coronavirus Disease 2019
(COVID-19); and find possible risk factors that are associated with
re-infection.

\textbf{Methods}

\emph{Study design and participants}

This prospective study was performed for a duration of six months from
May to September of 2020 on Coronavirus Disease 2019 (COVID-19) patients
who were admitted to the Imam Khomeini Referral Hospital, Tehran, Iran.

\emph{Eligibility Criteria}

Patients who had been diagnosed with COVID-19 (approved by a Real-Time
Polymerase Chain Reaction (RT-PCR) test of nasopharyngeal specimens) and
had been admitted based on the national criteria for hospitalization (a
sustained peripheral oxygen saturation of under 93\% and/or a
respiratory rate of over 30/minute or sustained nausea and vomiting and
severe weakness even with normal oxygen saturation), were brought into
the study upon discharge. The current national discharge criteria
dictate that prior to discharge patients must have at least two
consecutive afebrile days with a blood oxygen saturation of over 90\%,
oral intake without nausea, and improvement in weakness. The exclusion
criteria were a negative RT-PCR result or lack of documentation of a
positive result at the initial hospitalization.

\emph{Study initiation and follow-up sessions}

All information regarding patients' admission and epidemiological data
including age, sex, any past medical history and resent use of
immunocompromising medications were documented.

The first follow-up RT-PCR study was performed one month after the
initial positive RT-PCR test; at which point all patients had been
asymptomatic for at least 14 days. All patients who tested negative were
re-tested one and three months after the date of the first follow-up
RT-PCR test (Figure-1). A complete assessment of signs and symptoms
related to COVID-19 along with serology testing for anti-SARS-CoV-2 IgG
and IgM levels were also performed during the
3\textsuperscript{rd}follow-up visit.

\emph{RT-PCR}

A nasopharyngeal swab sample was obtained by a trained technician and
RNA was extracted with a Real Genomics viral nucleic acid extraction kit
(Cat.No.YVN50/YVN100). The Novel Coronavirus (2019-nCOV) Nucleic Acid
Diagnostic Kit (PCR-Fluorescence Probing), Sansure Biotech (S3102E),
made in Changsha, China was used for qualitative detection of the
ORF1ab and N genes of 2019-nCOV with a cycle threshold of less than 35
(Ct\textless{}35) for positive control and Ct\textgreater{}40 for
negative control at channel FAM, ROX and CY5 (internal control)
according to kit instructions. 

\emph{Antibodies}

A 5cc whole blood sample was drawn (without anticoagulants), and serum
was derived from the specimen using centrifugation (3000xg for 10
minutes). The Enzyme-linked Immunosorbent Assay (ELISA) method (Pishtaz
Teb SARS-CoV-2 IgM and IgG- Iran) was used to test the serum antibody
levels. The test was performed according to the manufacturer's
brochure. Results greater than 1.1 were considered positive and those
less than 0.9 as negative. Results within the mentioned range were
reported as borderline and the test was redone on a second, fresh serum
sample to confirm the initial results.

The manufacture reported diagnostic specifications of the test kits: 

Specificity: 97.30\% and Sensitivity: 79.40\% for SARS-COV-2-IGM kit. 

Specificity: 98.30\%\% and sensitivity: 91.10\% for SARS-COV-2-IGG kit.


\emph{Measurements and statistical analysis}

Data was analyzed using SPSS software V.22.0. Quantitative variables are
reported by mean and standard deviation (SD) and qualitative variables
are reported using frequency and percentage. Chi-square and Fisher's
exact tests were used to assess the statistical relationships between
categorical variables. The level of significance was set as P-value
\textless{} 0.05 for all analyses.

\emph{Ethical considerations}

Informed consent was obtained from participants and they were advised
that they can leave the study at any time point and this will not hinder
their current treatment or future visits to the hospital and the quality
of care they would receive. This study was conducted in accordance with
the 1964 Helsinki Declaration and approved by the Tehran University of
Medical Sciences ethics board committee (Ethics code:
IR.TUMS.VCR.REC.1399.076).

\textbf{Results}

\textbf{One month after the first positive RT-PCR (1st F/U)}

Overall, 202 discharged patients were contacted for follow-up laboratory
studies and 82 patients, including 27 women (33\%) and 55 men (64\%),
decided to participate in this study.

Patients had a median age of 47 ranging between 29 and 84 years old.
Table 1 shows the demographic description of participants. The first
RT-PCR results of 4 patients (4.87\%) were positive; who were
asymptomatic and not different from those who tested negative in terms
of the duration of the initial admission, the severity of the first
episode of the disease, presence and type of underlying diseases, and
recent history of using immunosuppressive drugs (P-value: 0.63, 0.57,
0.59, 0.61).

\textbf{Two months after the first positive RT-PCR (2nd F/U)}

One month after the first negative follow-up result, among the 78
patients who had tested negative, 44 patients (53.65\%) continued their
participation in the study. Additional data regarding patients who
tested positive are presented in Figure-1. The patients who tested
positive were not different in terms of the duration of the initial
admission, severity of the disease, and the need for ICU admission
(P-value: 0.69, 0.62, and 0.65).

\textbf{Four months after the first positive RT-PCR (3rd F/U)}

\textbf{A} mong the 41 patients who had tested negative, 32 patients
(39.02\%) continued their participation in the study. Four patients had
a positive RT-PCR result (12.5\%). None of the patients who tested
positive were symptomatic in previous studies; but now three of them
were mildly symptomatic; also, two patients had underlying medical
conditions (Figure-1). None of the patients who tested negative were
symptomatic. The underlying medical conditions, history of using
immunosuppressive drugs, initial disease severity, admission duration
and history of ICU admission in the first episode of infection were not
significantly different between patients who had a positive RT-PCR
result and those who did not (P-value: 0.67, 0.59, 0.64, 0.64, 0.71).

The IgM levels were \textless{}1.1 g/L in 30 (94\%) patients (negative);
and positive in 2 patients. The patients who tested positive were
asymptomatic and did not have a positive RT-PCR test in any of the
previous follow-up studies. The IgG levels were \textgreater{}1.1 g/L in
30 (94\%) patients (positive); and negative in 2 patients. The mean IgG
level was 7.94\selectlanguage{ngerman}±3.67 g/L and ranged between 1.48-14.5 g/L. The two
patients, who had a negative IgG test result, did not test positive in
any of the follow-up RT-PCR tests.

All patients recovered with no further complications and none of the
patients who re-tested positive required readmission or further medical
treatment.

\textbf{Discussion}

\textbf{Interpretations of a positive RT-PCR re-test}

Considering the high infection rate, few cases of reinfection with
SARS-CoV-2 have been reported. Often, for the diagnosis COVID-19 in a
recovered individual a positive RT-PCR test with or without accompanying
symptoms has been used. Although, in these cases different speculations
for interpretation of a re-positivity can be made; each of which will
have specific reverberations {[}21{]}.

In some studies, a positive RT-PCR in a recovering patient who tested
negative upon resolution of their symptoms, has been considered a strong
indicator of reinfection {[}10{]}. Although, we should consider the
possibility of a false result in either test {[}22{]}. A false positive
result can occur in a recovering patient; also a false negative result
shortly after subsidence of symptoms, followed by a correctly positive
test misleads physicians towards a re-infection diagnosis {[}23{]}. To
avoid this problem---as per WHO recommendations---in many regions the
treatment protocol for COVID-19 requires two consecutive negative RT-PCR
results prior to discharge {[}24, 25{]}.

Also, RT-PCR cannot distinguish between replication-competent viruses
and remnants of viral fragments that are expelled from a recovering
patient {[}26, 27{]}. Viral shedding from the respiratory tract during
the recovery period has been reported to last for as long as 12 weeks
after infection {[}28{]}; thus, a significant time-gap between the first
episode of infection and a positive RT-PCR test can clarify that the
patient has passed the viral shedding stage.

The first follow-up RT-PCR

As per national protocol, RT-PCR testing was not performed upon
discharge to confirm viral clearance. The first follow-up RT-PCR study
was performed 30 days after the first positive RT-PCR result in each
patient, which was after at least 14 days of being asymptomatic.
Considering the short interval between the onset of the disease and the
test, no recorded negative RT-PCR result at discharge, and the fact that
the patients who tested positive were asymptomatic, we believe that the
positive results show continued viral shedding rather than re-infection.
The four patients who tested positive were not significantly different
from others in terms of the severity of the initial illness, duration of
the first admission, age, or underlying conditions to suggest a risk
factor for a longer viral shedding period. Other studies have reported
that up to 14\% of recovering asymptomatic patients who tested negative
upon discharge, re-test positive {[}29{]}. Other studies have reported
patients who became symptomatic after a short symptom-free period. A
24-year-old health-worker became symptomatic and tested positive for
SARS-CoV-2 within 52 days of an initial symptomatic diagnosis of
COVID-19. Similar to our study, no confirmatory RT-PCR testing was
performed at discharge. Serum antibodies were not detected at the
beginning of the second symptomatic period, which can represent an
incomplete immune response that left the infection temporarily dormant,
only to be re-activated again; and although the patient was symptomatic
and had occupational contact with infected patients during her
symptom-free period, re-activation was considered more probable than
re-infection {[}30{]}. Similar studies have reported a return of mild or
even severe symptoms; but within a short time frame and without genetic
analysis of the infective pathogens, they were reported to be cases of
reactivation rather than re-infection {[}31-34{]}.

The second follow-up RT-PCR

Among the 44 patients who tested negative in the first test and
continued their cooperation with the study, 3 patients (6.81\%) tested
positive 60 days after the initial infection (one month after the first
negative RT-PCR test). These patients again were asymptomatic and the
interval between the initial infection and this positive test is not
significant enough to rule out viral shedding. Although, in case of
viral shedding and/or presence of remnant viral particles, we would have
expected to achieve a positive result in the previous test as well as
this one; but this dissonance can be explained in absence of
re-infection. Given the limited accuracy of RT-PCR, in case of a false
negative in the first follow-up test or a false positive in the second
test, continued shedding or complete recovery (respectively) can be
misdiagnosed as re-infection {[}35, 36{]}. On the other hand, studies
have shown that COVID-19 patients have a lower concentration of ACE2
monocyte expression---the endogenous entry receptor of SARS-CoV-2---and
researchers hypothesize that the virus can remain dormant in peripheral
blood mononuclear cells and cause a relapse after the respiratory system
has been cleared of the virus and patient has tested negative
{[}37-39{]}.

Positive RT-PCR tests in absence of significant symptoms in a recovering
patient has also been reported in many studies {[}18, 40{]}. In some
cases, symptoms were present, although less severe than the first
episode {[}41{]}. Our findings include asymptomatic and mildly
symptomatic patients who re-tested positive for SARS-CoV-2. None of our
cases had symptoms as or more severe than the first episode of
infection. Contrasting our results, the majority of other reports have
described more severe symptoms in patients who re-tested positive
{[}15-17, 42{]} and researchers have hypothesized that a selection bias
towards testing and confirming re-infection in symptomatic patients
{[}43{]} and/or a primed/heightened immune response upon the second
episode of infection can be the reason why most cases of re-infection
pertain to patients with intense symptoms upon second exposure and
infection {[}15, 42{]}.

An episode of re-infection can be proved by a positive viral culture
{[}21, 44{]} and/or genetic sequencing of the infective virus in both
episodes of infection to confirm that the second episode is caused by a
different clade of the virus. Tillett et al, reported a case of
re-infection in a 25-year-old male, who recovered from a
RT-PCR-confirmed episode of COVID-19, only to become symptomatic again
after a 30-day symptom-free period. Genetic sequencing showed distinct
genetic differences between the two specimen, indicating two separate
instances of infection with genetically different variants of the virus
{[}15{]}. Similar cases of re-infection with a genetically different
clade of the virus within 6 months of an original episode of COVID-19
have been reported {[}16-18{]}.

Unfortunately, genetic sequencing is not readily available and based on
CDC recommendations in absence of genetic proof of infection with a
different clade of the virus, a positive RT-PCR test that has been
obtained after the first 90 days of the onset of the initial infection
can be considered indicative of re-infection. Although, a positive
RT-PCR test after two consecutive negative results, especially if
accompanied by typical symptoms, can be defined as re-infection even
within the first 90 days of the first episode of infection {[}21{]}.

The third follow-up RT-PCR

According to the recommended CDC definitions {[}28{]}, re-infection is a
probable diagnosis for the three symptomatic patients who tested
positive in the last RT-PCR test; but it is less likely in case of the
one asymptomatic patient. The third RT-PCR screening was performed 120
days after the diagnosis of COVID-19 in 32 patients (who had tested
negative on both of the previous tests). Although, the symptoms could
have been caused by re-activation of dormant infection and release of
viruses from body reservoirs, they could also be caused by re-infection.
A similar process involving latent infection of cells followed by
transcription of viral genome has also been suggested, which would
result in reactivation of the virus from a latent to a lytic stage after
a symptom-free period, causing a resurgence of COVID-19 symptoms
{[}39{]}; but the long interval between the two positive RT-PCR results
makes re-activation an unlikely diagnosis {[}21{]}. These four patients
were not significantly different from those who tested negative in terms
of past medical history or severity of the initial episode of COVID-19.
Also, we did not find any risk factors that could help distinguish
patients who are more susceptible to re-infection from those who are
not.

\textbf{Humoral response}

Both insufficient and overactive immune responses have been reported in
COVID-19 patients {[}45{]}. Production of protective cytokines and
IFN-gamma, which is mediated by the CD4+ and CD8+ cells, plays an
important role in containing and resolution of the infection {[}46{]}.
The dynamics of the antibody response in COVID-19 patients is not
completely known; and different studies have reported different rates of
seroconversion. Zhao et al {[}47{]} and Liu et al {[}48{]} reported
seroconversion in all infected patients respectively by 39 and 14 days
after the onset of infection. Liu et al also reported that by the
60\textsuperscript{th} day IgM antibodies were undetectable in about
one-third of the patients and the IgG titers had decreased substantially
{[}48{]}. Based on another study, although recently discharged patients
have a high level of humoral immunity against the virus, antibodies
start to decreases within 2 to 3 months after the infection {[}13{]}. In
another study the seroconversion rate for IgG, IgM and IgA was
\textasciitilde{}90\% and most patients seroreverted within 75 days;
with IgG levels remaining detectable over 90 days after the symptom
onset in more than 99\% of patients {[}49{]}. Multiple studies have also
concluded that the humoral immunity against this virus could be
short-lived {[}50{]}. Contrasting these studies, our results showed that
94\% of patients were positive for neutralizing antibodies (IgG) 120
days after the onset of symptoms; which is in line with the results of
an Icelandic population study that reported a 91\% seropositivity four
months after the initial diagnosis of COVID-19 {[}6{]}. In evaluation of
the results of these studies, we should take into account the natural
process of the humoral response. In case of many other viral
infections---where seroconversion is sustained as seromaintenance and
immunity---we see a temporary decrease of antibody levels during the
first few months of infection/inoculation {[}51{]}, and since the
emergence of COVID-19 is recent, we cannot judge the humoral response to
this virus in long term and a rebound increase in antibody levels can be
expected {[}50{]}.

In our study, the four patients who had a positive result in RT-PCR
screening 120 days after the initial diagnosis of COVID-19, were also
positive for neutralizing antibodies; and although they theoretically
may have prevented a severe episode of re-infection and caused a lack of
any symptoms in one RT-PCR-positive patient, we cannot know for sure if
those levels are high enough to be completely protective {[}21{]}. In a
similar study Zhang et.al reported re-infection in 6 recovered patients
that was caused by viruses from lineages different from the first
infection. All these patients had varied levels of antibodies and they
concluded that presence and even maintenance of the humoral response
cannot rule out the possibility of re-infection {[}52{]}. We also
believe that the two patients who did not have sufficient levels of IgG
(\textless{}1.1 g/L), have been protected from an episode of
re-infection by a strong cellular immune response, even within an
epidemic situation.

Genetic sequencing can rule out a false positive in RT-PCR testing and
determine if the infection is caused by a different subclass of the
virus. In cases of re-infection with a different clade of the virus,
even protective levels of IgG may not be effective {[}21{]}. We
hypothesize that high levels of neutralizing antibodies do not make the
diagnosis of re-infection unlikely, unless there is genetic proof that
the positive RT-PCR results are related to the same strain of the virus
from the first episode; in which case re-activation/relapse would be a
more likely diagnosis.

\textbf{Limitations}

Some of the limitations of our study were that a considerable number of
participants dropped out during the study, this study was only performed
in one medical center, and that we could not perform genetic sequencing
to prove infection with a different clade of the virus, or cultures to
prove presence of replication-competent virus. The diagnosis of
re-infection in our study is based on RT-PCR results, typical symptoms
and the long interval between the two positive results. And, since we
did not measure the viral load in patients who tested positive, the
diagnosis of re-infection in our study is as certain as is the
specificity of the RT-PCR test. We were also unable to perform required
serology testing from the beginning of the study\textbf{.}

\textbf{Conclusion}

Based on current diagnostic guidelines, re-infection associated with
mild symptoms was detected in 3 out of 82 patients; barring possible
false positive results. We cannot confirm re-infection without positive
viral cultures. We also believe that the quantity of antibodies that are
produced against this virus can be sustained longer than the initial
studies suggest, although the protective abilities of these antibodies
against infection with the same or a different subtype of the virus
needs to be studied further. The emergence of new vaccines against this
virus, is a considerable achievement with limited guaranteed outcomes,
because the intricacies of the long-term immune response against this
virus is not fully known, and further studies on cases of supposed
re-infection are needed to clarify the probability and underlying risk
factors of re-infection.

\textbf{Declarations of interest:} none

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\textbf{Author contributions statement:}

Sara Sadr: Writing - Original Draft, Melika Arab Bafrani: Investigation,
Alireza Abdollahi: Conceptualization, Investigation, SeyedAhmad
SeyedAlinaghi: Formal analysis, Esmaeil, Mohammadnejad: Data Curation,
Roghieh Hossienzade: Investigation, Fereshteh Shahmari Golestan : Data
Curation, Zahra Ahmadinejad: Writing - Review \& Editing, Mohamadreza
Salehi: Supervision, Mohammad Javaherian: Investigation, Elahe kimyaee:
Methodology, Fatemeh Jafari: Data Curation, Fereshteh Ghiasvand: Project
administration and execution,

\textbf{Table 1-} Demographic data of participants\selectlanguage{english}
\begin{longtable}[]{@{}lllll@{}}
\toprule
& & \textbf{1\textsuperscript{st} F/U} & \textbf{2\textsuperscript{nd}
F/U} & \textbf{3\textsuperscript{rd} F/U}\tabularnewline
\midrule
\endhead
\textbf{Number of patients} & \textbf{Number of patients} & 82 & 44 &
32\tabularnewline
\textbf{Female} & \textbf{Female} & 25 & 16 & 12\tabularnewline
\textbf{Male} & \textbf{Male} & 57 & 28 & 20\tabularnewline
\textbf{Age} & \textbf{Age} & 52\selectlanguage{ngerman}±14 & 49\selectlanguage{ngerman}±16 & 47\selectlanguage{ngerman}±16\tabularnewline
\selectlanguage{english}\textbf{Underlying disease\textsuperscript{+}:} & \textbf{Underlying
disease\textsuperscript{+}:} & \textbf{Underlying
disease\textsuperscript{+}:} & \textbf{Underlying
disease\textsuperscript{+}:} & \textbf{Underlying
disease\textsuperscript{+}:}\tabularnewline
& \textbf{DM} & 12 & 9 & 5\tabularnewline
& \textbf{HTN} & 27 & 21 & 13\tabularnewline
& \textbf{Cardiovascular dis.} & 5 & 2 & 0\tabularnewline
& \textbf{Chronic pulmonary dis.} & 3 & 1 & 1\tabularnewline
& \textbf{Malignancies} & 2 & 0 & 0\tabularnewline
& \textbf{AIDS} & 1 & 1 & 0\tabularnewline
& \textbf{Smoker} & 11 & 8 & 7\tabularnewline
& \textbf{Recent use of immunosuppressive agents} & 1 & 0 &
0\tabularnewline
+ No patients in our study had a history of ESRD (End-Stage Renal
Disease), chronic liver disease, neurological disorders, morbid obesity,
transplantation or any other immune-compromising condition other than
AIDS. DM (Diabetes mellitus), HTN (Hypertension), AIDS (Acquired
immunodeficiency syndrome) & + No patients in our study had a history of
ESRD (End-Stage Renal Disease), chronic liver disease, neurological
disorders, morbid obesity, transplantation or any other
immune-compromising condition other than AIDS. DM (Diabetes mellitus),
HTN (Hypertension), AIDS (Acquired immunodeficiency syndrome) & + No
patients in our study had a history of ESRD (End-Stage Renal Disease),
chronic liver disease, neurological disorders, morbid obesity,
transplantation or any other immune-compromising condition other than
AIDS. DM (Diabetes mellitus), HTN (Hypertension), AIDS (Acquired
immunodeficiency syndrome) & + No patients in our study had a history of
ESRD (End-Stage Renal Disease), chronic liver disease, neurological
disorders, morbid obesity, transplantation or any other
immune-compromising condition other than AIDS. DM (Diabetes mellitus),
HTN (Hypertension), AIDS (Acquired immunodeficiency syndrome) & + No
patients in our study had a history of ESRD (End-Stage Renal Disease),
chronic liver disease, neurological disorders, morbid obesity,
transplantation or any other immune-compromising condition other than
AIDS. DM (Diabetes mellitus), HTN (Hypertension), AIDS (Acquired
immunodeficiency syndrome)\tabularnewline
\bottomrule
\end{longtable}

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