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\begin{document}

\title{On the trail of the novel coronavirus at the start of the pandemic in
the Lazio region: a retrospective research of SARS-CoV-2 in
nasopharyngeal swabs.}



\author[1]{Giuseppe Sberna}%
\author[1]{Eleonora Lalle}%
\author[1]{Licia Bordi}%
\author[1]{Emanuele Nicastri}%
\author[1]{Giuseppe Ippolito}%
\author[1]{Alessandra Amendola}%
\author[1]{Maria Capobianchi}%
\affil[1]{National Institute for Infectious Diseases  Lazzaro Spallanzani Institute for Hospitalization and Care Scientific}%


\vspace{-1em}



  
  \date{May 21, 2024}


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\selectlanguage{english}
\begin{abstract}
SARS-CoV-2 RNA was not detected in 219 nasal/oro-pharyngeal swabs,
analyzed for respiratory disease diagnosis at the National Institute for
Infectious Diseases ``Lazzaro Spallanzani'', from November 2019 to
January 2020; these results suggest lack of unrecognized SARS-CoV-2
circulation before the identification of the first two cases in Italy.%
\end{abstract}%



\sloppy


\textbf{On the trail of the novel coronavirus at the start of the
pandemic in the Lazio region: a retrospective research of SARS-CoV-2 in
nasopharyngeal swabs.}

Giuseppe Sberna\textsuperscript{1}, Eleonora Lalle\textsuperscript{1},
Licia Bordi\textsuperscript{1}, Emanuele Nicastri\textsuperscript{2},
Maria Rosaria Capobianchi\textsuperscript{1}, Giuseppe
Ippolito\textsuperscript{3}, Alessandra Amendola\textsuperscript{1*}

\textsuperscript{1} Laboratory of Virology, National Institute for
Infectious Diseases ``L. Spallanzani'' IRCCS, Rome, Italy.

\textsuperscript{2} Clinical Department, National Institute for
Infectious Diseases ``L. Spallanzani'' IRCCS, Rome, Italy.

\textsuperscript{3} Scientific Direction, National Institute for
Infectious Diseases ''L. Spallanzani'' IRCCS, Rome, Italy.

\textsuperscript{*}Corresponding Author:

\emph{Alessandra Amendola, PhD}

Laboratory of Virology

National Institute of Infectious Diseases ``L. Spallanzani''

Via Portuense 292, 00149, Rome, Italy

Tel: +390655170660

e-mail: alessandra.amendola@inmi.it

\textbf{Abstract}

SARS-CoV-2 RNA was not detected in 219 nasal/oro-pharyngeal swabs,
analyzed for respiratory disease diagnosis at the National Institute for
Infectious Diseases ``Lazzaro Spallanzani'', from November 2019 to
January 2020; these results suggest lack of unrecognized SARS-CoV-2
circulation before the identification of the first two cases in Italy.

The first cases of COVID-19 diagnosed in Italy, at the National
Institute for Infectious Diseases ``Lazzaro Spallanzani'' (INMI) in
Rome, were two Chinese travelers (on January 29, 2020) as reported to
the WHO European Region surveillance system {[}1, 2{]}. However, these
cases did not ignite local transmission of the virus; in fact, the first
case on SARS-CoV-2 infection acquired by local transmission in Italy
dates back to February 21, 2020 in Codogno (Lodi province), while in
Lazio region the first autochthonous case was diagnosed on March 1,
2020.

An interesting paper recently published {[}3{]} showed an oropharyngeal
swabs (OPS) sample resulted PCR positive for the SARS-CoV-2 RNA, among
samples collected for rubella and measles surveillance purpose, from
young patients in Milan (September 2019 - February 2020). The Authors
reported the earliest evidence of the presence of SARSCoV-2 RNA in
December 2019, dating back to about 3 months before the first COVID-19
case in Italy, anticipating the beginning of the outbreak in Lombardy
region to late autumn 2019 {[}3{]}, in agreement with other evidence of
early SARSCoV-2 spread in northern Italy {[}4{]}.

We conducted a similar investigation in Lazio region, by analysing 219
naso/oro-pharyngeal swabs (NPS) obtained from patients hospitalize at
INMI or in other hospitals of Lazio region for the presence of
respiratory symptoms. The demographic data of the study patients are
reported in Table 1. These specimens were collected from November 19,
2019 to January 27, 2020 and analyzed immediately after arrival in the
laboratory for the presence of non-SARS-CoV-2 respiratory pathogens,
using QIAstat-Dx Respiratory Panel (Qiagen s.r.l, USA). Hence, they were
frozen at -80\selectlanguage{ngerman}°C and never defrosted, until the SARS-CoV-2 RNA assay,
that was recently carried out for the purpose of this study, using
Aptima\textsuperscript{TM} SARS-CoV-2 (Hologic, Inc., CA) or Simplexa\selectlanguage{english}
COVID-19 Direct (DiaSorin Molecular LLC, CA) molecular assays. Out of
219 NPS, 71 resulted positive for non-SARS-CoV-2 respiratory pathogens
and no one was positive for the SARS-CoV-2 RNA (Table 1).

Although limited to a small number of samples, these data are well
representative of epidemiological situation in Lazio region during the
considered period, as they reflect the frequency of respiratory
pathogens observed in the same period of the previous year, with the
exception of Influenza A cases, accordingly to the epidemic seasonal
trend (https://w3.iss.it/site/RMI/influnet/pagine/stagioni.aspx) (Table
1).

In addition, it is worth to note that our results are perfectly aligned
with the data of wastewater-based epidemiology, reported by Italian
Istituto Superiore di Sanita, which highlight the presence of SARS-CoV-2
RNA in Rome sewage since March 31, 2020 {[}5{]}.

Therefore, the results obtained with the molecular test for SARS-CoV-2
performed retrospectively on the samples collected in the previous three
months before the first case diagnosed at INMI, support the start the
SARS-CoV-2 circulation in Lazio region shortly before March 2020,
confirming the epidemiological data so far available.

\textbf{Table 1.} Description of tested naso/oro-pharyngeal swabs.\selectlanguage{english}
\begin{longtable}[]{@{}lllll@{}}
\toprule
Period & Period & November 2018 - January 2019 & November 2019 - January
2020 & \emph{p*}\tabularnewline
\midrule
\endhead
N. Samples & N. Samples & 250 & 219 &\tabularnewline
Age {[}mean (min -- max) years{]} & Age {[}mean (min -- max) years{]} &
55 (1 -- 90) & 54 (0 -- 99) &\tabularnewline
Gender {[}\%F - \%M{]} & Gender {[}\%F - \%M{]} & 37.2 -- 62.8 & 42.5 --
57.5 & \emph{0.2570}\tabularnewline
Pathogens {[}N.Positive (\%){]} & Pathogens {[}N.Positive (\%){]} & &
&\tabularnewline
Influenza A viruses: & Influenza A viruses: & 67 (26.8) & 26 (11.9) &
\emph{0.0005}\tabularnewline
& H1N1 pdm09 & 55 (22.0) & 22 (10.0) & \emph{0.0045}\tabularnewline
& H3 & 8 (3.2) & 4 (1.8) & \emph{0.3940}\tabularnewline
Rhino/Enterovirus & Rhino/Enterovirus & 21 (8.4) & 14 (6.4) &
\emph{0.4820}\tabularnewline
Respiratory Syncytial Virus & Respiratory Syncytial Virus & 23 (9.2) &
12 (5.5) & \emph{0.1580}\tabularnewline
Coronavirus non-SARS-CoV-2 & Coronavirus non-SARS-CoV-2 & 8 (3.2) & 4
(1.8) & \emph{0.3940}\tabularnewline
\emph{Mycoplasma Pneumoniae} & \emph{Mycoplasma Pneumoniae} & 1 (0.4) &
9 (4.1) & \emph{0.0070}\tabularnewline
Others & Others & 13 (5.2) & 12 (5.5) & \emph{1.0000}\tabularnewline
Coinfections & Coinfections & 20 (8.0) & 6 (2.7) &
\emph{0.0140}\tabularnewline
\bottomrule
\end{longtable}

*statistical evaluation based on Fisher exact test
(https://www.scistat.com/statisticaltests/fisher.php).\textbf{}

\textbf{References}

\begin{enumerate}
\tightlist
\item
  Spiteri G, Fielding J, Diercke M, Campese C, Enouf V, Gaymard A, et
  al. First cases of coronavirus disease 2019 (COVID-19) in the WHO
  European Region, 24 January to 21 February 2020. Euro Surveill. 2020
  Mar;25(9):2000178. doi: 10.2807/1560-7917.ES.2020.25.9.2000178. PMID:
  32156327; PMCID: PMC7068164.
\item
  Capobianchi MR, Rueca M, Messina F, Giombini E, Carletti F, Colavita
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  of COVID-19 in Italy. Clin Microbiol Infect. 2020 Jul;26(7):954-956.
  doi: 10.1016/j.cmi.2020.03.025. Epub 2020 Mar 27. PMID: 32229288;
  PMCID: PMC7118617.
\item
  Amendola A, Bianchi S, Gori M, Colzani D, Canuti M, Borghi E, et al.
  Evidence of SARS-CoV-2 RNA in an Oropharyngeal Swab Specimen, Milan,
  Italy, Early December 2019. Emerg Infect Dis. 2021;27(2):648-650.
\item
  La Rosa G, Mancini P, Bonanno Ferraro G, Veneri C, Iaconelli M,
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\item
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  Bonadonna L, et al. First detection of SARS-CoV-2 in untreated
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\end{enumerate}

\textbf{Author's contribution}

Amendola A, Sberna G: analysed data, performed laboratory testing, wrote
manuscript; Lalle E, Bordi L: designed the study, read the manuscript,
performed laboratory testing; Capobianchi MR: discussed results, read
and revised manuscript; Ippolito G, Nicastri E: discussed results.

\textbf{Funding}

This research was supported by funds to National Institute for
Infectious Diseases `Lazzaro Spallanzani' IRCCS from Ministero della
Salute (Ricerca Corrente, linea 1; COVID-2020 - 12371817), the European
Commission -- Horizon 2020 (EU project 101003544 -- CoNVat; EU project
101003551 -- EXSCALATE4CoV; EU project 101005111 - DECISION; EU project
101005075 - KRONO) and the European Virus Archive -- GLOBAL (grants no.
653316 and no. 871029).

\textbf{Competing interests}

None declared.

\textbf{Ethical approval}

Ethical approval was not required as residual samples were used and
patients' data remained anonymous.

\textbf{Acknowledgments}

We gratefully acknowledge the INMI's Laboratories staff.

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