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\begin{document}

\title{NATURAL HISTORY OF POSTNATAL HUMAN CYTOMEGALOVIRUS INFECTION}



\author[1]{Piera d’Angelo}%
\author[1]{PAOLA ZELINI}%
\author[1]{Federica Zavaglio}%
\author[1]{Stefania Piccini}%
\author[2]{Daniela Cirasola}%
\author[1]{Alessia Arossa}%
\author[1]{Arsenio Spinillo}%
\author[1]{Daniele Lilleri}%
\author[1]{Fausto Baldanti}%
\affil[1]{Fondazione IRCCS Policlinico San Matteo}%
\affil[2]{IRCCS Humanitas Research Hospital}%


\vspace{-1em}



  
  \date{May 24, 2024}


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\selectlanguage{english}
\begin{abstract}
Postnatal cytomegalovirus (HCMV) infection is well characterized in
preterm infants, where it can lead to severe symptomatic infection. We
analyzed the rate and route of transmission of postnatal HCMV infections
in full-term babies during the first year of life. A cohort of 120 HCMV
seropositive mothers and their 122 newborns were tested after delivery
for HCMV DNA shedding in different bodily fluids. Postnatal HCMV
infection was defined as the detection of \textgreater{}2.5\selectlanguage{ngerman}×10
\textsuperscript{2} HCMV-DNA copies/mL in infants' saliva swabs.
Maternal neutralizing antibody serum titer, HCMV specific T-cell
response, and HCMV glycoprotein B (gB) IgG on breastmilk were analyzed.
HCMV shedding was detected in 67 of 120 mothers (55.8\%), and 20 of 122
infants (16.4\%) developed HCMV infection within the first three months
of life. Six additional infants were infected during the first year, for
a postnatal infection rate of 21.3\%. Viral shedding was more frequent
in breastmilk than saliva, urine and vaginal secretions, and the mothers
of infected infants showed higher levels of HCMV-DNA in milk. No
association was found between the antibody levels in serum or milk and
maternal viral shedding, whereas a slightly lower frequency of
HCMV-specific CD4 \textsuperscript{+} T-cells with long-term memory
phenotype was observed in women with HCM-DNA-positive milk. About one
out of five infants develop HCMV infection within the first year of
life. Breastmilk appears the major route of transmission of the
infection, maternal saliva have a minor role whereas the role of vaginal
secretions is negligible.%
\end{abstract}\selectlanguage{ngerman}%



\sloppy


\textbf{INTRODUCTION}

Human cytomegalovirus (HCMV) is the leading cause of congenital
infections, occurring in 0.5-2\% of pregnancies, with about 11\% of
live-born symptomatic infected infants. Congenital HCMV infection is
more frequent after a primary HCMV infection during pregnancy than after
non-primary infection (reactivation or
reinfection).\textsuperscript{1}Moreover, HCMV seems to be the principal
cause of postnatal infection in the world. In fact, despite previous
immunity, seropositive women may reactivate the infection in mammary
gland and subsequently excretes the virus in the
breastmilk.\textsuperscript{2,3} In addition, HCMV shedding at delivery
was observed in multiple bodily fluids,\textsuperscript{4,5}among which
vaginal secretions and saliva may represent potential routes of HCMV
transmission to the newborn. It has been seen that HCMV reactivation
during the first 3 months after birth occur in \textgreater{}95\% of
seropositive mothers.\textsuperscript{6} However, in full term babies,
transmission via breastfeeding is usually asymptomatic due to the
protection given by maternal IgG antibodies transferred through the
placenta, in particular after the 28\textsuperscript{th} week of
gestation. However, it can lead to severe symptomatic HCMV-infection in
preterm infants, in particular if the newborn is a very low birth weight
or if is less than 32 weeks of gestational age.\textsuperscript{6,7}
Moreover reactivation in the genital tract can be considered a source of
transmission for HCMV infection during delivery. Postnatal transmission
is well characterized in pre-term newborns at high risk of symptomatic
infection, while not much is known of the natural history of postnatal
transmission in full-term, healthy newborns. Infants with postnatal HCMV
infection usually have normal cognitive development and the infection
does not affect the neurodevelopment during the first 6 years of live.
However, a study conducted in the United Kingdom assessed that HCMV
infection was associated with suboptimal attention control at 8 years of
age.\textsuperscript{8,9}

This article will focus on the frequency HCMV of non-primary infection
(NPI, namely HCMV reactivation) occurring post-delivery in a group of
mothers of full-term infants and we will describe the source (in
particular in breastmilk \emph{vs} saliva and genital secretions) and
the rate of transmission to their infants, during the first 6-12 months
of life.

\textbf{MATERIALS AND METHODS}

\emph{Study design and participants}

Between June 2017 and June 2022, 120 HCMV-seropositive women and their
infants were enrolled at Fondazione IRCCS Policlinico San Matteo (Pavia,
Italy). Maternal saliva swab, urine, vaginal swab and milk (when
possible), and saliva from infants were collected after delivery (T0;
median days since delivery 2, interquartile range -IQR- 1-3 days), and
after three-six months (T1; median days since delivery 94, IQR 71-115
days). In a subgroup of 38 mother-infant pairs with a HCMV-DNA positive
sample at T0 and/or T1, we also analyzed a subsequent time point, T2
(T2; median days since delivery 307, IQR 231-417 days), before the
admission of the infant to a day care center.

\emph{HCMV-DNA quantification}

Samples were tested for HCMV-DNA using Artus CMV PCR kit, (Qiagen,
Hilden, Germany) after DNA extraction with EZ1 DSP virus kit (Qiagen)
using EZ1 Advanced XL (Qiagen). We considered a cut-off for HCMV
infection in infants of 2.5\selectlanguage{ngerman}×10\textsuperscript{2} HCMV-DNA copies/mL in
saliva swabs. This cut-off was based on a previous study on congenital
HCMV infection, in which among 45 newborns with HCMV-DNA positive
saliva, the infection was confirmed only in cases with a level of DNA
above 2.5×10\textsuperscript{2} copies/mL.\textsuperscript{10}

\emph{T-cell response}

HCMV-specific T-cell response was analyzed in 15 women as previously
described.\textsuperscript{11,12} After the identification of memory
T-cells by exclusion of naïve T-cells
(CD45RA\textsuperscript{+}CCR7\textsuperscript{+}),
CD4\textsuperscript{+} and CD8\textsuperscript{+} IFN-\selectlanguage{greek}γ \selectlanguage{english}producing
T-cells were determined, and among them the percentages of cells
IL-7R\textsuperscript{+} (long-term memory, LTM) or
IL-2\textsuperscript{+} were calculated.

\emph{ELISA}

HCMV glycoprotein B (gB) IgG on breastmilk was tested by ELISA as
previously described, with some minor
changes.\textsuperscript{13}Briefly, half-area 96-well microplates were
coated with 25 \selectlanguage{greek}µ\selectlanguage{english}l/well of 3\selectlanguage{greek}µ\selectlanguage{english}g/ml gB (GSK plc, Brentford, United Kingdom)
for 1 hour. Following incubation, plates were blocked for 2 hours with a
solution of 80.5\% PBS, 4\% whey protein, 15\% goat serum, and 0.5\%
Tween-20; then breastmilk (1:3 in PBS), was added to wells for 1.5
hours. After washing, the plates were incubated with horseradish
peroxidase-labeled goat IgG to human IgG (SouthernBiotech, Birmingham,
AL, USA) for 45' (1:4000 in PBS-skimmed milk 1\%). Finally, plates were
incubated for 25' with 30 mg/mL orthophenylendiamine (Sigma-Aldrich, St.
Louis, MO, USA) before the addition of 4N sulphuric acid. Plates were
read at 450nm.

\emph{Neutralization assay}

HCMV neutralizing antibody (VR1814 for epithelial cells and AD169 for
fibroblasts) were tested as reported. \textsuperscript{14} The serum
dilution inhibiting virus infectivity by 50\% was considered the 50\%
neutralizing antibody titer.

\subsection*{\texorpdfstring{\emph{Statistical
analysis}}{Statistical analysis}}

{\label{statistical-analysis}}

Categorical variables were described as count and percentage and
compared using Fisher's exact test. For numerical variables, the median
value with IQR was reported and they were compared with the Mann-Whitney
U-test. Comparison between more than two groups was performed using the
Kruskall-Wallis test and Dunn's post test with correction for multiple
comparisons. The Spearman R with its 95\% confidence interval (95\%CI)
was calculated for correlation analysis. A p-value \textless{}0.05 was
considered statistically significant. Analyses were performed with
GraphPad Prism version 8.3.0 (GraphPad software La Jolla, CA, USA).

\textbf{RESULTS}

\emph{Maternal HCMV reactivation after delivery and rate of postnatal
HCMV infection in newborns.}

Overall, 67/120 (55.8\%) women developed a NPI post-delivery, in
particular 24 (20\%) at T0 and 43 (35.8\%) subsequently at T1. HCMV-DNA
was detected in 8/120 (6.7\%) saliva swabs, in 13/117 (11.1\%) vaginal
swabs, in 6/116 (5\%) urine and in 7/9 (77.8\%) milk samples at T0. At
T1 we found HCMV-DNA in 11/120 (9.0\%) saliva swabs, 15/120 (12.5\%)
vaginal swabs, in 8/119 (6.7\%) urine and in 49/83 (59.0\%) milk samples
(Fig 1.A). None of the 122 infants (two sets of twins) was positive for
HCMV-DNA in saliva after delivery, thus excluding a congenital HCMV
infection. Subsequently, 20/122 (16.4\%) infants developed HCMV
infection after birth at T1 (Fig 1.B). Infants with HCMV infection did
not shown severe symptoms. Among maternal bodily fluids, HCMV-DNA load
was higher in milk at T0 and at T1 compared with all the other samples
of the mothers at both time points (p\textless{}0.001), with a median
value of 2,551 (IQR: 6-26,467) copies/mL and 105 (IQR: 0-1,410)
copies/mL, respectively (Fig 1.C).

To verify whether some characteristic of the infants recorded at
delivery could be at the basis for a susceptibility to the HCMV
infection, we compared them between infants with and without infection.
No statistically significant difference was found in infants with HCMV
infection compared with those without infection regarding the
gestational age at birth, the type of delivery, the sex and the weight
and length at birth, instead we observed a significant but small
difference in the head circumference (p=0.03). (Table 1).

\textbf{Table 1.} Characteristics of infants with and without HCMV
infection within the first three months of life\selectlanguage{english}
\begin{longtable}[]{@{}llll@{}}
\toprule
& Infected infants (n=20) & Non-infected infants (n=102) & \emph{p
Value}\tabularnewline
\midrule
\endhead
Weeks of gestation at birth (median; IQR) & 40; 39-40 & 40; 39-40 &
\emph{0.83}\tabularnewline
Type of delivery: & & & \emph{0.76}\tabularnewline
\emph{- Natural delivery (number; \%)} & 16; 80\% & 82; 80\% &
\emph{}\tabularnewline
\emph{- C-section (number; \%)} & 4; 20\% & 18; 18\% &
\emph{}\tabularnewline
Sex: & & & \emph{0.15}\tabularnewline
\emph{- Male (number; \%)} & 7; 35\% & 55; 54\% & \emph{}\tabularnewline
\emph{- Female (number; \%)} & 13; 65\% & 47; 46\% &
\emph{}\tabularnewline
Weight \emph{grams} (median; IQR) & 3290; 3079-3378 & 3295; 3009-3513 &
\emph{0.65}\tabularnewline
Length \emph{cm} (median; IQR) & 51; 50-52 & 51; 50-52 &
\emph{0.43}\tabularnewline
Head circumference \emph{cm} (median; IQR) & 34; 33-34 & 35; 34-35 &
0.03\tabularnewline
Mother with NPI (number; \%) & 18; 90\% & 50; 50\% &
\textless{}0.01\tabularnewline
\bottomrule
\end{longtable}

NPI, non-primary infection; IQR, interquartile range

A subgroup of 38 mother-infant pairs, in which a positive HCMV-DNA
sample was found in T0 and/or T1, was followed for a longer period,
until the admission of the infant to the daycare center, to verify
whether infants of HCMV shedding mothers may be infected at a later time
after birth but before entering in contact with other potential sources
of HCMV (i.e. other infants attending the daycare center). Of the 38
infants, 12 had a primary infection diagnosed at T1 and were still
shedding HCMV at T2 (Fig. 1D), while in six infants the infection was
diagnosed subsequently at T2, leading to a cumulative number of 26/122
infants (Fig. 1.B) with HCMV infection at one year of age (21.3\%). Of
note, two mothers of the six infants were still breastfeeding and the
milk samples were positive for HCMV-DNA.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image1/image1}

\end{center}
\end{figure}

\textbf{Figure 1.} Frequency of HCMV-DNA detection (A) and HCMV viral
loads in saliva, vaginal swabs, urine and milk of mothers (C). Frequency
of HCMV infection in infants at T1 and T2 (B) and viral loads in saliva
(blue dots represents infants who were infected later at T2) (D). HCMV,
human cytomegalovirus

\emph{Source of postnatal HCMV infection}

In order to understand the source of transmission of the virus to the
infants, we compared HCMV shedding in the following bodily fluids
between the transmitting and non-transmitting mothers: i) vaginal swabs
at delivery (in women who had a natural childbirth); ii) milk at T1 and
T2 (milk at T0 was not analyzed here due to the small number of samples
collected); and iii) saliva swabs at the three time points. No
significant difference were found in the viral load of vaginal swabs
(p=0.65) and of saliva swabs (p=0.80) at delivery of transmitting
mothers compared to non-transmitting ones (Fig 2.A and B). Conversely,
at T1, we observed a significant difference in the viral load of both
breastmilk (p\textless{}0.01) and saliva swab (p=0.01) a T2 (Fig. 2.C
and D). In addition, viral load in both breastmilk and saliva swabs of
transmitting mothers was significantly higher (p=0.03 and p=0.04,
respectively), compared with non-transmitting mothers, also at T2 (Fig.
2.E and F). A significant although weak correlation between viral load
in infants' saliva swabs and in saliva and milk of mothers was found at
T1 (p\textless{}0.01, R= 0.31; 95\%CI= 0,14-0,47 and p\textless{}0.01,
R= 0.43; 95\%CI= 0.23-0.59, respectively).\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image2/image2}

\end{center}
\end{figure}

\textbf{Figure 2.} HCMV viral load in vaginal swabs (A) and in saliva
swabs at delivery (B), in milk (C) and saliva swabs (D) at T1 and in
milk (E) and saliva swabs (F) at T2, compared between transmitting
mothers and non-transmitting mothers. HCMV, human cytomegalovirus

Of the 120 women analyzed, 92 (76.7\%) breastfed their infant: in 85
(92.4\%) of them milk was collected, while for seven (7.6\%) women
breastmilk was not available due to difficulties during the collection.
These seven mother-infant pairs were excluded from the following
analysis (two of these infants developed HCMV infection). The other 28
(23.3\%) women used the formula. Overall, 113 mother-infant pairs, among
which 24 infants developed HCMV infection, were analyzed. HCMV-DNA was
found in milk of 52/85 (61.2\%) breastfeeding mothers at T0 or T1. Among
the 52 mothers with HCMV-DNA in milk, 21 (40.4\%) had an infant with
HCMV infection (12 of these 21 women were not shedding the virus in the
saliva). One (3.6\%) of the 33 breastfeeding mothers with
HCMV-DNA-negative milk transmitted the infection to the infant, and she
was shedding the virus through the saliva. Only 2/28 (7.1\%) infants fed
with formula had HCMV infection, and in this case one mother showed the
highest level of HCMV-DNA in saliva swab of all the cohort
(\textgreater{}5000 copies/mL), while the other did not shed HCMV-DNA in
the saliva (Fig. 3). It was not possible to define a potential source of
infection for this infant, who might have been infected by other family
members. Despite the significant difference of viral load in saliva
swabs of transmitting mothers compared to non-transmitting mothers, milk
seems to be the major route of transmission: all but three infants with
HCMV infection had breastfeeding mothers with presence of HCMV-DNA in
milk, while maternal saliva was negative for HCMV-DNA for 13 infected
infants.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image3/image3}

\end{center}
\end{figure}

\textbf{Figure 3.} Route of transmission. Comparison between
breastfeeding and non-breastfeeding mothers with and without
HCMV-DNA-positive saliva swabs. Blue slides indicate mothers with
HCMV-DNA-positive saliva swabs, squared pattern infants with HCMV
infection and blank pattern infants without infection. HCMV, human
cytomegalovirus

\emph{Maternal HCMV-specific immune response and HCMV reactivation in
breastmilk.}

HCMV-specific T-cells and antibody levels in blood and milk were
determined in a subset of 8 women with and 7 women without HCMV-DNA in
milk in order to investigate a potential association between maternal
HCMV-specific immunity and HCMV shedding in milk. HCMV-specific LTM
T-cells were determined, analyzing the CD4\textsuperscript{+} and
CD8\textsuperscript{+} T-cells that expressed IL-7R, and produced IL-2
(Fig. 4). The percentage of
CD4\textsuperscript{+}IL-7R\textsuperscript{+} (p=0.04) and
CD8\textsuperscript{+}IL-7R\textsuperscript{+} (p=0.09) showed a trend
toward lower values in mothers shedding HCMV in the milk.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image4/image4}

\end{center}
\end{figure}

\textbf{Figure 4.} Percentages of HCMV-specific
CD4\textsuperscript{+}and CD8\textsuperscript{+} T-cells with LTM
(long-term memory) and producing IL-2 in women with and without HCMV-DNA
in milk. HCMV, human cytomegalovirus; IL7-R interleukin 7 receptor

In addition, the neutralizing activity of antibody against the infection
of epithelial cells and fibroblast was compared between mothers with
HCMV-DNA positive and negative milk. Neutralizing antibody titer against
the infection of epithelial cells (p=0.03) or fibroblasts (p=0.07) was
significantly higher in mothers who shed HCMV-DNA in milk (Fig.5).\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image5/image5}

\end{center}
\end{figure}

\textbf{Figure 5.} Serum antibody titers neutralizing HCMV (VR1814)
infection of ARPE-19 cells (A) and HCMV (AD-169) infection of HELF cells
(B) in women with and without HCMV-DNA in milk\textbf{.} HCMV, human
cytomegalovirus; HELF, human lung fibroblast; ARPE-19, human retinal
pigmented epithelial.

Previous studies showed anti-gB IgG titer higher in infants without HCMV
infection, moreover, the anti-gB IgG titer was moderately associated
with delayed HCMV acquisition.\textsuperscript{15} Here the presence of
anti-gB IgG was tested in breastmilk, due to the lack of infants' serum,
to verify whether also IgG in milk could be protective against the
acquisition of the infection in infants. Comparison between milk of
transmitting and non-transmitting mother did not show a significant
difference (p=0.84) (Figure 6.A). In addition, no significant difference
was found while comparing HCMV-DNA positive or negative milk (p=0.35)
(Figure 6.B).\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image6/image6}

\end{center}
\end{figure}

\textbf{Figure 6.} Comparison of anti-gB IgG in milk between
transmitting and non-transmitting mothers (A) and between HCMV-DNA
positive and negative milk. HCMV, human cytomegalovirus; gB,
glycoprotein B

\textbf{DISCUSSION}

In this study, the frequency of postnatal HCMV infection was evaluated
in a group of healthy infants born to HCMV-seropositive mothers. After a
median time of three months after birth, 16.4\% of enrolled infants
(20/122) had HCMV infection, whereas within the first year of life,
before admission to daycare centers, the rate of infection increased to
21.3\% (26/122). Of the 120 mothers, 55.8\% had a NPI after delivery,
according to detection of HCMV shedding in bodily fluids. In particular,
HCMV-DNA was detected more frequently and at higher levels in breastmilk
(59\% of women in whom breastmilk was collected shed the virus in this
compartment), whereas HCMV shedding in the other bodily fluid was
detected in a lower percentage of women (12.5\% women shed HCMV in
vaginal secretions, 9\% in saliva and 6.7\% in urine).

It is known that HCMV reactivation occur during pregnancy, in particular
in the genital tract.\textsuperscript{4} Previous studies show that
perinatal transmission can occur due to viral elimination through
genital secretions,\textsuperscript{16,17} but here no difference was
seen in vaginal swabs HCMV-DNA load between transmitting and
non-transmitting mothers, thus excluding the genital tract as a
significant source of perinatal infection. Breastmilk transmission has
been intensively studied in low-birth weight and preterm infants with a
rate of acquisition of HCMV infection ranging from 5.7\% to
60\%.\textsuperscript{17} Breastmilk seems to be the major route of
transmission of the infection in the group of infants analyzed in this
study, since viral load was higher in the milk of mothers of infected
infants, and a significant correlation between viral load in maternal
milk and infants' saliva was observed. Despite a significant difference
of viral load was also found in saliva swabs of transmitting mothers
compared to non-transmitting mothers, we could speculate that saliva
does not represent a major source of infection for the infants. In fact,
all but three of the infected infants were breastfed by mothers shedding
HCMV in milk, whereas the mothers of 15/26 infected infants did not shed
HCMV in saliva. Nevertheless, maternal saliva could be considered as a
potential minor route of transmission, and the mother of one of the two
infected child who was not breastfed showed the highest HCMV-DNA load in
saliva (\textgreater{}5000 copies/mL). Viral shedding in saliva of
mothers of infected infants could also be the effect of a local
infection caused by an infant-to-mother transmission after primary
infection of the child, as already described in a similar cohort of
mother-infant pairs in Uganda.\textsuperscript{18}

The prevalence of infection increases with age, and a previous study
conducted in Italy showed that HCMV infects 56\% of the population in
the first 5 years of life.\textsuperscript{16} Another study conducted
in the USA showed that 31\% infants of 0-47 months were
HCMV-seropositive.\textsuperscript{19} Analyzing the rate of infection
at 1 year of life, we observed that 21.3\% (26/122) of infants born to
seropositive women develop a HCMV infection, although most of them are
infected within the first 3-4 months. It is likely that HCMV infection
during infancy occurs in two periods: a first period is within the first
months after delivery, through contact with breastmilk, whereas a second
period occurs after contact with other infants, especially when
attending daycare centers: a French study reported a 51.9\% prevalence
of HCMV shedding in infants attending daycare
centers.\textsuperscript{20}

In our study, we observed that about 1 out of three infants of
HCMV-shedding mothers developed HCMV infection. Different
characteristics of the infants and of the pregnancy have been compared
between infants with or without HCMV infection, looking for a
susceptibility to the infection. Except for a small difference found in
the head circumference, no difference in gestational age, weight and
length at birth has been found, in line with the fact that our
population is made of full term, healthy newborns.

Previous studies suggested a protective role of high level of anti-gB
IgG in infants' serum for postnatal HCMV
infection.\textsuperscript{15}We could not analyze anti-HCMV antibody
levels in infants' serum, but we investigated whether the presence of
anti-HCMV antibody (namely anti-gB IgG) in breastmilk was associated
with prevention of HCMV transmission. However, the levels in milk of
these antibodies were not different between transmitting and
non-transmitting mothers. Moreover, no significant difference was found
comparing HCMV-DNA-positive milk with HCMV-DNA-negative milk, thus
excluding any role in controlling local virus replication.

Conversely, the systemic HCMV-specific immune response appeared
different between women who reactivated or not HCMV in breastmilk. In
particular, women shedding HCMV in milk had a lower frequency of
HCMV-specific T-cells with a LTM phenotype, and a higher titer of
neutralizing antibodies. Higher level of neutralizing antibodies against
epithelial HCMV infection was observed in a population of solid-organ
transplanted host, compared with primary HCMV infection along with a
viral load peak much higher.\textsuperscript{21} Whether these
characteristics indicate an inconsistent immune control, which may be at
the basis of the HCMV reactivation from latency, or rather are a
consequence of a greater immune stimulation induced by the HCMV
reactivation itself, could be investigated in future studies. In line
with this data, we also observed a higher anti-gB IgG level in pregnant
women with compared to those without HCMV
reactivation.\textsuperscript{4}

The strength of this study is the comprehensive analysis of HCMV
shedding in maternal bodily fluids and its relationship with postnatal
HCMV infection of the newborns. Its major limitation is the lack of
long-term follow-up for the infants whose mother were not shedding HCMV
after delivery, which may have underestimated the actual rate of HCMV
infection within one year of age. However, it is unlikely that a
significant number of infections have been missed because of this
design.

In conclusion, about 20\% of infants born to HCMV seropositive women
develop HCMV infection within the first year of life (mainly within the
first three months). The major source of infection is breastmilk, in
which about 60\% of seropositive women shed HCMV, and one out of three
infants receiving HCMV-positive milk develop HCMV infection. Conversely,
maternal saliva represent a minor source of infection whereas the role
of genital tract secretion is negligible. This data could be informative
for future vaccination strategies, when considering the vaccination of
infants.\textsuperscript{22,23}

\textbf{Funding:} This study was supported in part by a research grant
(no. MISP 59300) from Investigator-Initiated Studies Program of Merck
Sharp \& Dohme Corp. The opinions expressed in this paper are those of
the authors and do not necessarily represent those of Merck Sharp \&
Dohme Corp. The study was also funded by Fondazione Regionale per la
Ricerca Biomedica (Grant no. FRRB 2015-043).

\textbf{Ethics Approval Statement}

The study was approved by the Ethics Committee and Fondazione IRCCS
Policlinico San Matteo Institutional Review Board (Procedure n\selectlanguage{ngerman}°
P-20170007596 and P-20190073479).

\textbf{Informed Consent Statement:} Informed consent was obtained from
all subjects involved in the study.

\textbf{Conflict of Interest}

The authors declare no conflict of interest.

\textbf{Data Availability Statement}

The data that support the findings of this study are available from the
corresponding authors upon reasonable request.

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