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Epidemiology, prenatal diagnosis
and maternal treatment
Tiziana Lazzarotto
O.
O. U.
U. of
of Clinical
Clinical Microbiology,
Microbiology, Laboratory
Laboratory of
of Virology
Virology
St.
St. Orsola
Orsola Malpighi
Malpighi University
University Hospital,
Hospital,
University
University of
of Bologna,
Bologna, Bologna,
Bologna, Italy.
Italy.
The overall birth prevalence
CONGENITAL
CMV
INFECTION
of congenital CMV
infection
is 0.64%
PRIMARY
INFECTION
reactivationNON PRIMARY ((reactivationreinfection
reinfection))
INFECTION
PREGNANT WOMEN
Transplacenta
Transplacentall route
route
FOETUS
FOETUS
Lazzarotto
Tiziana
Risk of congenital CMV infection following
PRIMARY and NON PRIMARY infection
of the mother during pregnancy
Effect of gestational age on transmission in utero.
Stagno S. et al. JAMA 1986
Congenital infection
the rate of transmission
40-50% Primary infection ∼70%
Primary infection
1
Lazzarotto
Tiziana
20
10
Gestational Age, wk
38-42
Risk of congenital CMV infection following
PRIMARY and NON PRIMARY infection
of the mother during pregnancy
Effect of gestational age on transmission in utero.
Stagno S. et al. JAMA 1986
Congenital infection
the rate of transmission
1 - 2%
40-50% Primary infection ∼70%
Non primary
Primaryinfection
infection
1
Lazzarotto
Tiziana
20
10
Gestational Age, wk
38-42
Risk of congenital CMV infection following
PRIMARY and NON PRIMARY infection
of the mother during pregnancy
Effect of gestational age on transmission in utero disease in the offspring – and severe sequelae
Stagno S. et al. JAMA 1986
Congenital infection
the rate of transmission
Symptoms at birth
1-2%
Primary – Risk ∼10-15%
Non primary - Risk ∼2-5%
1
Lazzarotto
Tiziana
10
20
Gestational Age, wk
38-42
Risk of congenital CMV infection following
PRIMARY and NON PRIMARY infection
of the mother during pregnancy
Effect of gestational age on transmission in utero disease in the offspring – and severe sequelae
Stagno S. et al. JAMA 1986
Congenital infection
the rate of transmission
Significant
handicaps
Symptoms
at birth
1-2%
– Risk
∼10-15%hearing loss
mental Primary
retardation
- bilateral
NonRisk
primary
- Risk
∼25%
primary
∼30-40%
; non
primary
Risk ∼1-4%
1
Lazzarotto
Tiziana
10
20
Gestational Age, wk
38-42
CMV Infection and Pregnancy
TOPICS
Lazzarotto
Tiziana
1.
Epidemiology
2.
Diagnosis of maternal infection
3.
Diagnosis and (prognosis)
of fetal infection
4.
Maternal treatment
CMV Infection and Pregnancy
TOPIC
1.
Epidemiology
2.
Diagnosis of maternal infection
3.
Diagnosis and (prognosis)
of fetal infection
4.
Maternal treatment
Lazzarotto
Tiziana
EPIDEMIOLOGY
Prevalence
Prevalence of
of seropositivity
seropositivity to
to CMV
CMV in
in various
various regions
regions of
of the
the world
world
Lazzarotto
Tiziana
Country
Continent
%
Ireland
Europe
30
Germany
Europe
42
France
Europe
51
Melbourne
Australia
57
Alabama
North America
59
Italy
Europe
68
Greece
Europe
78
Spain
Europe
79
Argentina
South America
81
Brazil
South America
84
Israel
Asia
85
Taiwan
Asia
91
India
Asia
99
Uganda
Africa
100
Female
CMV seroprevalence in Italy
15 - 45 years
∼20.000
90.00%
80.00%
70.00%
60.00%
50.00%
15 - 19
anni
%pos 59.02%
Vecchia L et al. 2002
Lazzarotto
Tiziana
20 - 24
anni
71.98%
25 - 29
anni
69.21%
30 - 34
anni
68.78%
35 - 39
anni
74.59%
40 - 44
anni
85.42%
CMV seroprevalence in Italy
%pos
% neg
100,00%
80,00%
60,00%
40,00%
20,00%
0,00%
gravide
0 - 6 mesi 7 - 12 mesi
% neg
27,30%
22,66%
63,72%
%pos
72,70%
76,40%
33,63%
Vecchia L et al. 2002
Between
Between 30-40%
30-40% of
of children
children at
at
11 year
year of
of age
age are
are CMV-infected
CMV-infected
Lazzarotto
Tiziana
EPIDEMIOLOGY
The management of CMV infection during
pregnancy depends on CMV seroprevalence
Study
% Mothers
CMV Ab +
% Cong CMV
Infection
London, UK, 1983
25
0.24
Birmingham, USA, 1986(middle SES)
36
0.53
Hamilton, Canada, 1980
52
0.4
Aarhus-Viborg, Denmark, 1979
52
0.4
Sao Paulo, Brazil, 1985 (middle SES)
67
0.46
Sal Paulo, Brazil, 1985 (lower SES)
84
0.98
Birmingham, USA, 1986 (lower SES)
77
1.25
Seoul, Korea, 1991
96
1.2
Santiago, Chile, 1978
98
1.7
Abidjan, Ivory Coast, 1978
100
2.4
Lazzarotto
Tiziana
The higher the rate of maternal seroprevalence,
the higher the rate of congenital CMV infection.
Epidemiology CONCLUSION
Primary maternal infections have a much
greater clinical impact on the fetus than
non primary infection
Stagno et al 1986 - 2002
Maternal
Maternal reinfection
reinfection by
by new
new strains
strains of
of CMV
CMV is
is aa major
major
source
source of
of congenital
congenital infection
infection in
in populations
populations with
with
seroprevalence
seroprevalence approaching
approaching 100%
100% (Yamamoto
(Yamamoto et
et al.
al. AJOG,
AJOG,
2010).
2010).
Severe
Severe symptoms
symptoms are
are almost
almost exclusively
exclusively due
due to
to
exogenous
exogenous reinfections
reinfections with
with aa different
different strain
strain of
of CMV
CMV
rather
rather than
than the
the reactivation
reactivation of
of latent
latent virus
virus (Congenital
(Congenital
CMV
CMV Conference,
Conference, San
San Francisco
Francisco USA
USA 2012).
2012).
Lazzarotto
Tiziana
CMV Infection and Pregnancy
TOPIC
1.
Epidemiology
2.
Diagnosis of maternal infection
3.
Diagnosis and (prognosis)
of fetal infection
1.
Prevention/treatment
Lazzarotto
Tiziana
Diagnosis of maternal CMV infection
Serological diagnosis
reliable
Detection of IgG AND IgM
Lazzarotto
Tiziana
Diagnosis of CMV infection
The serological reaction employing
antibodies linked to a tracer enzymatic or
fluorescent or chemiluminescent.
Screening tests
EIA (ELISA), MEIA, CMIA, ELFA ,CLIA
EIA (Enzyme Immuno Assay) o ELISA (Enzyme-LinkedImmunoSorbent-Assay)
MEIA (Microparticle Enzyme Immunoassay)
Immunoassay
CMIA (Chemiluminescent Microparticle Immunoassay)
Immunoassay
ELFA (Enzyme-linked fluorescent assay)
CLIA (chemiluminescence immunoassay)
immunoassay
Lazzarotto
Tiziana
Serological diagnosis
is reliable
Diagnosis of primary CMV infection in pregnant women
Categories
Period
What does this mean?
What should be done
IgG –
IgM-
just before pregnancy
or
during pregnancy
Non immune woman
High risk of acquiring
primary infection
Information on hygiene
and behavioral measures
IgG +
IgM-
before ≤ 16 WG
Past infection
No further diagnostic
investigation is required
IgG –
IgM+
before or during
pregnancy
acute phase of primary
infection ??
or
IgM false positive
result ??
Serological testing
performed in the same
lab
after 10-15 days
IgG +
IgM+
before ≤ 16 WG
Active infection?
Primary infection?
Non primary ?
Advanced diagnosis →
Avidity IgG test
Lazzarotto
Tiziana
Diagnosis of primary maternal CMV infection
Why in this case would there be problems
in the interpretation of IgM
positive results?
Lazzarotto
Tiziana
Problems in the interpretation of specific CMV
-IgM
CMV-IgM
1. Virus
-specific IgM may persist unchanged for
Virus-specific
months after natural infection
2. Unspecific positive results may arise in subjects
with other viral infections (B19 Virus, EBV, etc.)
or autoimmune diseases
3. False positive results may also be caused by
laboratory methods.
4. IgM test will not distinguish between primary
and non
-primary infection, and not distinguish
non-primary
acute or recent or late phase of infection.
Lazzarotto
Tiziana
IgM cannot replace
a seroconversion
Can we do better
?
better?
Advanced diagnosis
Lazzarotto
Tiziana
AVIDITY TEST
Determination of the IgG
-CMV avidity
IgG-CMV
CMV-IgG maturation occurs between 18-20 weeks
70
HIGH
HIGH
Avidity index (%)
60
50
40
moderate
moderate
30
LOW
LOW
20
10
Lazzarotto et al. Clin Diagn
Lab Immunol,
Immunol, 1999.
0
0
5
10
15
20
25
30
35
Weeks after beginning of symptoms
Sensitivity
ting the
Sensitivity identification
identification of
of pregnant
pregnant women
women at
at risk
risk of
of transmit
transmitting
the virus
virus to
to fetus
fetus
before 16
-18 weeks gestation →
16-18
after 21
-22 weeks gestation →
21-22
sens = 100%
sens = 60%
Lazzarotto et al. Viral Immunol 2000
Lazzarotto
Tiziana
A high avidity index during the first 12
-16 weeks gestation
12-16
could be considered as a good indicator of past infection.
Ability
Ability of
of the
the IgG
IgG avidity
avidity assays
assays to
to detect
detect
and
and to
to exclude
exclude aa recent
recent primary
primary CMV
CMV infection
infection
Method
Before
Lazzarotto et al 1997 commercial 16-18 weeks
Sens (%)
92.8
Spec (%)
85.7
20 weeks
nd
100
Grangeot-Keros et al commercial
2001
17 weeks
nd
100
Bodéus et al 2001-
12 weeks
100
98
Revello et al 2004
commercial
commercial
12 weeks
92.8
84.7
Lagrou et al 2009
commercial
12 weeks
100
98
Enders et al 2000
in-house
Avidity & CMV Nt-Ab
Lazzarotto
Tiziana
Serum/plasma samples from primarily infected women
obtained at different weeks after beginning of infection.
Figure
Figure 1:
1: Kinetic
Kinetic of
of LIAISON
LIAISON 
 XL
XL CMV
CMV IgG-CLIA
IgG-CLIA avidity
avidity
y = 0.1382x + 8.3448
LIAISON XL CMV IgG avidity Index
70
2
R = 0.6739
HIGH avidity
60
50
40
30
MODERATE avidity
20
LOW avidity
10
0
0
50
100
150
200
250
days after onset of CMV primary infection
Lazzarotto T et al 2015, 5th International Congenital CMV Conference
Lazzarotto
Tiziana
300
Determination of the CMV - IgG avidity
Attention !
Interpreting test results is very important because
serological tests vary from one laboratory to another
so
the method used and its reference values must be
carefully assessed.
Lazzarotto
Tiziana
Attention !
Commercial kits for determination of the CMV - IgG avidity
Range and interpretation of results (according
’s instructions)
manufacturer
(according to
to manufacturer’
manufacturer’s
instructions)
AI
AI
AI
Kit 1
<50%
low
50 to 60 %
grey zone
>60%
high
Kit 2
<0.4
low
0.4 to 0.65
moderate
>0.65
high
Kit 3
<0.4
low
0.4 to 0.55
grey zone
>0.55
high
Kit 4
<0.2
low
0.2 to 0.3
moderate
>0.3
high
Kit 5
<30%
low
30 to 40%
borderline
>40%
high
Kit 6
<40%
low
40 to 60%
equivocal range
>60%
high
Kit 7
<35%
low
35 to 45%
moderate
>45%
high
Kit 8
<25%
low
25 to 45%
moderate
>45%
high
Kit 9
<45%
low
45 to 55%
grey zone
>55%
high
Lazzarotto
Tiziana
Roche
Roche Symposium
Symposium
CMV
CMV IgG
IgG avidity:
avidity: value
value
and
and limits
limits
Liliane
-Keros
Liliane Grangeot
Grangeot-Keros
((Clamart,
Clamart, France)
France)
The value of CMV IgG avidity
helps to interpret a positive result for CMV IgM
IgM..
Attention !
The limits of CMV IgG avidity
avidity::
1) the kinetics of CMV IgG avidity depend on the kit used
used,,
2) with some of these kits
kits,, the results may depend on CMV
IgG concentration
concentration..
Lazzarotto
Tiziana
CLINICAL
CASE
2013
-year-old woman
2013 -- aa 35
35-year-old
woman in
in her
her second
second pregnancy
pregnancy
Screening
Screening tests
tests
Neg < 6.0
2013
CMV-IgG
6≤x<15
9 weeks
15.00 UA/mL Equivocal 6≤
of gestation Weak Positive Pos ≥ 15
CMV-IgM
6.34 Index
Positive
Advanced
Advanced diagnosis
diagnosis
10 weeks
CMV-IgG
of gestation 26.10 UA/mL
Positive
CMV-IgM
10.22 Index
Positive
CMV-IgG
IB
Positive
CMV-IgM
IB
Positive
CMV IgG avidity
0.43 (high AI)
FALSE
Undetectable
Neg < 0.85
Equivocal 0.85≤
0.85≤x<1
Pos ≥ 1
CMV-DNA
Real Time PCR
Whole blood
4.215 copies/mL
Comment:
Comment: the
the very
very low
low titer
titer of
of IgG
IgG anti
anti CMV
CMV does
does not
not allow
allow an
an accurate
accurate
evaluation
evaluation of
of the
the IgG
IgG avidity
avidity test
test resulting
resulting undetectable.
undetectable.
ACUTE PHASE OF PRIMARY CMV INFECTION
Lazzarotto
Tiziana
2015
Lazzarotto
Tiziana
NATURAL HISTORY OF HUMAN CYTOMEGALOVIRUS
PRIMARY INFECTION
SALIVA
-DNA POSITIVE
CMV
SALIVA CMVCMV-DNA
POSITIVE
Neg
++
URINE
-DNA POSITIVE
CMV
URINE CMVCMV-DNA
POSITIVE
++
Neg
++
CMV
CMV DNAemia
DNAemia POSITIVE
POSITIVE
0
0
77
14
14
21
21
days
days
28
28
22
months
months
Time
Time after
after onset
onset of
of CMV
CMV infection
infection
Lazzarotto
Tiziana
44
66
Neg
88
Pregnant women with primary CMV infection
enrolled at the moment of serological diagnosis
Real Time PCR
n. of patients
total
n. of positive
patients
sensitivity
Whole blood
41
26
63.4%
saliva
41
32
78.0%
urine
41
25
60.9%
Pregnant women with non-primary CMV infection
enrolled at the moment of serological diagnosis
Real Time PCR
n. of patients
total
n. of positive
patients
sensitivity
Whole blood
27
5
18.6%
saliva
27
13
48.1%
urine
27
12
44.4%
Lazzarotto 2014
Conclusion - primary CMV infection
Serological
Serological
diagnosis
diagnosis
Virological
Virological
diagnosis
diagnosis
Antibodies
Antibodies
DNA
DNA
IgG and IgM with
screening tests
<12 WG
Saliva – Real Time PCR
Avidity IgG test
<16 WG
Urine - Real Time PCR
Whole blood
- Real Time PCR
C
AN BE
CAN
BE RELIABLE
RELIABLE
RELIABLE
RELIABLE
VIROLOGICAL
VIROLOGICAL DIAGNOSIS
DIAGNOSIS
1.
1. can
can support
support the
the serological
serological diagnosis
diagnosis of
of primary
primary CMV
CMV infection
infection
2.
2. plays
plays aa role
role in
in the
the diagnosis
diagnosis of
of non
non primary
primary CMV
CMV infection
infection
Lazzarotto
Tiziana
Diagnosis of non primary CMV infection
not always feasible
1.
IgG + IgM + high avidity ≤ 16WG
Absence of CMV in biological fluids
High risk of non
-primary CMV infection
non-primary
2.
IgG + IgM - high avidity ≤ 16WG
Presence of CMV in biological fluids
Non
-primary CMV infection
Non-primary
3.
IgG
+ and IgM
+ and high avidity ≤≤16WG
16WG
IgG+
IgM+
Presence of CMV in urine/saliva/blood (Real
(Real time
time PCR)
PCR)
Non
-primary CMV
Non-primary
CMV infection
infection
Attention !
Lazzarotto
Tiziana
No optimal diagnostic method
CMV Infection and Pregnancy
TOPIC
Lazzarotto
Tiziana
1.
Epidemiology
2.
Diagnosis of maternal infection
3.
Diagnosis and (prognosis) of fetal
infection
4.
Maternal treatment
CONGENITAL CMV INFECTION
Prenatal diagnosis
Invasive prenatal
diagnosis
Amniocentesis
Non invasive
prenatal diagnosis
+
Ultrasound
examination
+
COUNSELLING
When should prenatal diagnosis be performed
?
performed?
-8 weeks from the onset of maternal infection
after 66-8
CMV
-8 weeks
CMV is
is aa slow
slow replication
replication virus
virus and
and 66-8
weeks is
is the
the time
time required
required
from
from maternal
maternal infection
infection to
to virus
virus detection
detection in
in AF
AF
-21 weeks gestation
amniocentesis is performed between 20
20-21
Fetus
Fetus excretes
excretes CMV
CMV via
via urine
urine into
into the
the AF
AF and
and aa sufficient
sufficient amount
amount ooff fetal
fetal
diuresis
-21 WG
diuresis is
is produced
produced only
only after
after 20
20-21
WG
Lazzarotto
Tiziana
Amniocentesis: 20
-21 weeks gestation
20-21
Maternal
Maternal primary
primary
infection
infection
(the
(the first
first half
half of
of gestation)
gestation)
Observation
Observation of
of ultrasound
ultrasound
abnormalities
abnormalities
Which tests?
Virus isolation (shell vial)
Viral DNA by Real Time PCR
Lazzarotto
Tiziana
Virus isolation and Real Time PCR in 796 samples of amniotic
fluids of primarily infected women and congenital CMV infection
CONGENITAL INFECTION
Virus
isolation
Real Time
PCR
Copies/
Copies/mL
Yes
No
Total
Pos
140
0
140
Neg
49
607
656
Total
189
468
796
Pos
150
0
150
Neg
39
607
646
Total
189
607
796
SNS
%
SPE
%
PPV NPV
%
%
74.0
100
100
92.5
79.4
100
100
94.0
(Lazzarotto
(Lazzarotto TT et
et al.
al. CMV
CMV San
San Francisco
Francisco 2012)
2012)
PCR
PCR is
is more
more sensitive
sensitive than
than virus
virus isolation
isolation and
and the
the specificity
specificity ooff the
the two
two tests
tests is
is the
the same.
same.
Among
Among the
the 646
646 cases
cases with
with negative
negative result:
result: No
No congenital
congenital CMV
CMV infection
infection was
was identified
identified in
in
607
607 cases,
cases, while
while 39
39 newborns
newborns were
were infected
infected but
but were
were asymptomatic
asymptomatic at
at birth
birth and
and during
during
subsequent
subsequent monitoring
monitoring (NPV
(NPV 94%)
94%)
Lazzarotto
Tiziana
Literature reports since 2000.
Sensitivity, specificity, and predictive values
of PCR tests for CMV detection in amniotic fluid
Publication
year
SENS
%
SPE
%
PPV
%
NPV
%
Goegebuer et al.
2008
85
100
nd
nd
Revello et al.
Revello et al.
2004
2002
90.2
92.7
100
100
100
100
90.4
92.7
Enders et al.
2001
81.1
97.9
93.8
93.1
Gouarin et al.
2001
72
96.9
91.3
89
Azam et al.
2000
71
100
nd
nd
Liesnard et al.
2000
80
100
nd
nd
Author
nd:
nd: not done
Lazzarotto
Tiziana
Fetal CMV infection - counseling after prenatal diagnosis
AF
Virus
isolation
AF
DNA
PCR
Ultrasound
findings
OUTCOME - FETUSES
-
-
-
NO infection (∼94%) or
asymptomatic infection (∼6%)
+
YES infection (100%)
Low viral load
asymptomatic infection (100%)
(<1000 copies/ml)
copies/ml)
+
+
+
YES infection (100%)
High viral load
YES severe infection (100%)
(>105 copies/ml)
copies/ml)
+
+
High viral load
(>105 copies/ml)
copies/ml)
Lazzarotto
Tiziana
YES infection (100%)
symptomatic or asymptomatic
infection ?????
Under evaluation
Lazzarotto Tiziana, UO di Microbiologia,
Università di Bologna
-
CMV prenatal diagnosis
Why is it important to offer
a comprehensive (invasive and non invasive)
prenatal diagnosis
?
diagnosis?
Lazzarotto
Tiziana
It
It is
is currently
currently accepted
accepted that
that ultrasound
ultrasound examinations
examinations do
do not
not
identify
identify more
more than
than ∼∼20%
20% of
of infected
infected fetuses
fetuses and
and the
the sensibility
sensibility is
is
further
further reduced
reduced if
if the
the ultrasound
ultrasound is
is performed
performed when
when the
the state
state of
of
the
the CMV
CMV fetal
fetal infection
infection is
is unknown.
unknown.
Guerra, et al.
2008
Out of 600 pregnant
women who underwent
ultrasound
examinations with
complete follow up,
there were 51 abnormal
ultrasound findings.
From these 51 findings,
only 23 fetuses were
actually infected.
SENS
SPEC
PPV
NPV
Lazzarotto
Tiziana
14.9%
93.7%
45.0%
76.1%
Diagnosis of fetal CMV infection
20
-21 weeks
20-21
weeks gestation
gestation
Prenatal diagnosis is reliable
Amniotic fluid is the most appropriate material
for diagnosis of CMV fetal infection
Fetal blood does not give any additional diagnostic
value because the tests used are not sensitive
enough to detect the virus.
It also carries a high risk of fetal demise and
therefore should not be performed.
Lazzarotto
Tiziana
Is cordocentesis useful for prognostic
purposes in fetuses with CMV infection?
Prognosis of fetal CMV infection
20
-21 weeks
20-21
weeks gestation
gestation
The
The level
level of
of specific
specific IgM
IgM and
and viral
viral blood
blood load
load is
is not
not correlated
correlated
with
with aa poor
poor outcome.
outcome. ItIt has
has been
been proposed
proposed that
that platelet
platelet count
count
gives
gives aa better
better indication.
indication.
(Benoist
(Benoist et
et al.
al. AJOG
AJOG 2008)
2008)
The
The determination
determination of
of multiple
multiple markers
markers (haematological,
(haematological,
biochemical
biochemical and
and virological
virological markers)
markers) in
in fetal
fetal blood
blood following
following
virus
virus detection
detection in
in AF,
AF, is
is predictive
predictive of
of perinatal
perinatal outcome
outcome in
in
fetuses
fetuses with
with CMV
CMV infection.
infection.
(Fabbri
(Fabbri et
et al.
al. BJOG
BJOG 2011).
2011).
Lazzarotto
Tiziana
CMV
-IgM index and CMV load in fetal blood
CMV-IgM
of primarily infected women and
congenital CMV infection
Zavattoni
Zavattoni et
et al.
al. JJ Med
Med Virol
Virol 2014
2014
with
symptoms
Lazzarotto
Tiziana
without
symptoms
with
symptoms
without
symptoms
Study
Study population
population
Lazzarotto Tiziana, UO di Microbiologia,
Università di Bologna
15 pregnant women at 20-21 WG with diagnosis of CMV fetal
infection.
In all cases: amniotic fluids were positive for viral isolation and
qPCR with a high viral load (> 10^5 copies / mL).
At the moment of amniocentesis all pregnant women underwent
ultrasound examinations.
As negative control we studied 4 CMV-seronegative pregnant
women at 20-21 WG.
Study
Study design
design
- After elective pregnancy termination, 2 mL of fetal blood
were collected from the umbilical cord.
- Virological, haematological and biochemical markers in
fetal blood were studied.
This
This study
study (SA43GABR)
(SA43GABR) was
was approved
approved by
by the
the Ethical
Ethical Committee
Committee of
of St.
St. Orsola-Malpighi
Orsola-Malpighi General
General Hospital,
Hospital, Bologna.
Bologna.
19 pregnant women
RESULTS
RESULTS
electively
electively terminated
terminated their
their
pregnancy
pregnancy at
at 20-21
20-21 WG
WG
15 CMV infected fetuses
4 fetuses non CMV infected
histological
histological
evaluation
evaluation
12 fetuses
3 fetuses
CMV-pos
CMV-pos brain
brain (IHC)
(IHC)
CMV-neg
CMV-neg brain
brain (IHC)
(IHC)
brain
brain histological
histological
damage
damage
no
no brain
brain histological
histological
damage
damage
11 fetus
fetus cardiac
cardiac malformations
malformations
22 spina
spina bifida
bifida
11 trisomy
trisomy 21
21
CMV
CMV antigen
antigen expression
expression and
and inflammatory
inflammatory
response
response were
were studied
studied in
in all
all fetal
fetal tissues
tissues
using
immunohistochemical
staining
using
immunohistochemical
staining
procedures.
procedures.
Gabrielli
Gabrielli et
et al.
al. JJ Clin
Clin Virol
Virol 2009
2009
Gabrielli
et
al.
CMI
2012
Gabrielli et al. CMI 2012
Lazzarotto Tiziana, UO di Microbiologia,
Università di Bologna
Prognosis of fetal CMV infection
Case
#
ULTRASOUND at 20-21 WG
1
Negative
Brain damage
3
Negative
Brain damage
4
Negative
Brain damage
12 CMV
12
Negative
Brain damage
18
Negative
Brain damage
23
Negative
Brain damage
POSITIVE
BRAINS
(IHC)
AUTOPSY
0
Microcephaly
Brain damage
2
Cerebral periventricular echogenicity,
hyperechogenic bowel
Brain damage
16
Microcephaly, intraventricular septa,
hyperechogenic bowel
Brain damage
19
Cerebral ventriculomegaly
Brain damage
20
Hyperechogenic bowel
Brain damage
24
Cerebral periventricular echogenicity
Brain damage
7
Negative
with
histological
damage
Negative
3 CMV
8
Negative
Negative
NEGATIVE
BRAINS
(IHC)
9
Negative
Negative
without
histological
damage
Findings
Findings detected
detected by
by
ultrasound
and
ultrasound
and
correlation
correlation with
with or
or
without
without histological
histological
brain
brain damage
damage in
in the
the
15
15 fetuses
fetuses studied
studied
6/12
6/12 fetuses
fetuses with
with
histological
histological brain
brain
damage
damage had
had aa
pathological
pathological US
US
(50%).
(50%).
Sens
Sens:: 50%
50%
Spec:
Spec: 100%
100%
PPV:
PPV: 100%
100%
NPV:
NPV: 34%
34%
US
US examinations
examinations were
were performed
performed by
by one
one experienced
experienced ultrasonographer
ultrasonographer
working
working in
in S.
S. Orsola-Malpighi
Orsola-Malpighi Hospital
Hospital in
in Bologna,
Bologna, Italy
Italy (Dr.
(Dr. G.
G. Simonazzi).
Simonazzi).
Lazzarotto
Tiziana
Prognosis of fetal CMV infection
Fetal
-21 weeks
Fetal blood
blood samples
samples taken
taken at
at 20
20-21
weeks gestation
gestation
Serological
Serological markers
markers detected
detected in
in fetal
fetal plasma
plasma
of
-infected fetuses
of CMV
CMV-infected
fetuses and
and correlation
correlation with
with fetal
fetal brain
brain damage
damage
VIDAS®
ToRC panel
12 fetuses with
brain damage
3 fetuses without
brain damage
CMV IgM +
5
0
CMV IgM -
7
3
P
0.245
Fisher’s exact test
When
When comparing
comparing the
the blood
blood of
of infected
infected fetuses
fetuses with
with and
and without
without
brain
brain damage,
damage, we
we observed
observed no
no statistical
statistical difference
difference in
in
detection
detection of
of CMV
CMV specific
specific IgM
IgM antibodies.
antibodies.
Lazzarotto
Tiziana
Prognosis of fetal CMV infection
Fetal
-21 weeks
Fetal blood
blood samples
samples taken
taken at
at 20
20-21
weeks gestation
gestation
Virological
Virological markers
markers detected
detected in
in fetal
fetal whole
whole blood
blood
of
-infected fetuses
of CMV
CMV-infected
fetuses and
and correlation
correlation with
with fetal
fetal brain
brain damage
damage
Median values (range)
HCMV ELITe
MGB kit
12 fetuses with
brain damage
3 fetuses without
brain damage
Real time PCR
104,408
(1,470 – 1,993,500)
1,428
(0 – 16,335)
P
0.02
Wilcoxon test
On
On the
the contrary,
contrary, when
when comparing
comparing the
the blood
blood of
of infected
infected fetuses
fetuses
with
with and
and without
without brain
brain damage,
damage, we
we observed
observed aa statistical
statistical
difference
difference in
in median
median values
values of
of CMV-DNAemia
CMV-DNAemia levels.
levels.
Lazzarotto
Tiziana
Prognosis of fetal CMV infection
Fetal
-21 weeks
Fetal blood
blood samples
samples taken
taken at
at 20
20-21
weeks gestation
gestation
Virological
Virological markers
markers detected
detected in
in fetal
fetal whole
whole blood
blood
of
-infected fetuses
of CMV
CMV-infected
fetuses and
and correlation
correlation with
with fetal
fetal brain
brain damage
damage
107
106
105
104
103
102
10
without brain damage
with brain damage
CMV-infected fetuses
Lazzarotto
Tiziana
CMV DNA load in fetal blood samples is
significantly higher in fetuses with brain damage.
Prognosis of fetal CMV infection
Fetal
-21 weeks
Fetal blood
blood samples
samples taken
taken at
at 20
20-21
weeks gestation
gestation
Median values (range)
Fetal blood
parameters
Median values (range)
12 fetuses
with brain damage
3 fetuses
without brain damage
P value
3 fetuses
no CMV-infected
White blood cells
9.05x10^3/µL (2.6-16.4)
8.1x10^3/µL (3.8-10.1)
NS
10.1x10^3/µL (2.9-11.5)
Erythroblasts
10.2x10^3/µL (3.4-32.8)
1.8x10^3/µL (0.6-12.0)
NS
4.6x10^3/µL (0.2-56.4)
11.8 G/dL (8.0-12.5)
11.7 G/dL (11.0-11.7)
NS
8.5 G/dL (7.7-11.5)
3 469/mmc (666-8582)
3 334/mmc (1801-3818)
NS
0.6 (0.2-2.3)
0.8 (0.7-1.4)
NS
3.5 (2.7-6.1)
1 821/mmc (112-6012)
1 429/mmc (240-1530)
NS
1 079/mmc (104-1256)
ß2- Microglobulin
18.9 ng/mL (9.7-39.0)
7.7 ng/mL (4.1-9.7)
0.02
5.25 ng/mL (4.8-6.3)*
Platelet count
93x10^3/µL (18-161)
265x10^3/µL (126-315)
0.04
145x10^3/µL (126-154)
Hemoglobin
T lymphocytes
CD4/CD8
Natural killer
* statistical values obtained from 4 uninfected fetuses
1 719/mmc (1632-4814)
Wilcoxon
Wilcoxon test
test
The parameters that showed a statistically
significant difference between the two groups are
the ß
2-microglobulin and the platelet count .
ß2-microglobulin
Lazzarotto
Tiziana
Prognosis of fetal CMV infection
Fetal
-21 weeks
Fetal blood
blood samples
samples taken
taken 20
20-21
weeks gestation
gestation
Prognostic
Prognostic markers
markers in
in compliance
compliance of
of severe
severe fetal
fetal CMV
CMV disease
disease
1)
2- microglobulin
1) ß
ß2microglobulin >> 11
11 ng/mL
ng/mL
2)
µL
2) Platelet
Platelet count
count ≤≤ 110x10^3
110x10^3 //µL
3)
mL (Zavattoni
3) CMV
CMV DNAemia
DNAemia levels
levels >> 5.000
5.000 copies/
copies/mL
(Zavattoni 2014)
2014)
Markers
Agreement
YES
NO
total
Lazzarotto
Tiziana
9 fetuses with
brain damage
3 fetuses without
brain damage total
4
1
5
5
2
6
9
3
12
SENS
%
44
SPE
%
PPV
%
NPV
%
67
80
29
Lazzarotto Tiziana, UO di Microbiologia,
Università di Bologna
Therefore, prenatal counseling based on the compliance
of these specific markers can be misleading.
CLINICAL
CASE #1
Primary
Primary infection
infection in
in the
the first
first trimester
trimester of
of pregnancy
pregnancy
Prenatal
Prenatal diagnosis
diagnosis at
at 20
20 weeks
weeks of
of gestation
gestation
Amniotic fluid
Virological testings
Results
Virus isolation (shell vial)
Positive
Real Time PCR
Positive
Ultrasound Findings and
Cerebral Magnetic resonance
Fetal blood
ß2- Microglobulin
Platelet count
negative
cut-off
Results
(> 11 ng/mL)
14.6 ng/mL
(≤ 110x10^3 /µL)
CMV DNAemia levels
5.100.000 copies/mL ●
(> 5.000 copies/mL)
●
161x10^3 /µL
1.470 copies/mL
Autopsy: symptomatic fetus had CMV positive brain with severe
histological brain damage and necrosis of cerebral tissue.
Lazzarotto
Tiziana
CLINICAL
CASE #7
Primary
Primary infection
infection in
in the
the first
first trimester
trimester of
of pregnancy
pregnancy
Prenatal
Prenatal diagnosis
diagnosis at
at 21
21 weeks
weeks of
of gestation
gestation
Amniotic fluid
Virological testings
Results
Virus isolation (shell vial)
Positive
Real Time PCR
Positive
Ultrasound Findings and
Cerebral Magnetic resonance
negative
Fetal blood
ß2- Microglobulin
Platelet count
CMV DNAemia levels
cut-off
(> 11 ng/mL)
(≤ 110x10^3 /µL)
(> 5.000 copies/mL)
3.072.000 copies/mL
●
Results
9.7 ng/mL
126x10^3 /µL
16.335 copies/mL ●
Autopsy: CMV infected fetus without brain damage
Lazzarotto
Tiziana
STUDY LIMITATION
• Limited sample size does not allow firm conclusions.
However these data indicate the need to take further
caution when:
1) offering cordocentesis for fetal prognosis of CMV
infection;
2) evaluating blood markers for fetal prognosis of CMV
infection.
Lazzarotto
Tiziana
CONCLUSIONS
Further studies in a larger number of symptomatic and
asymptomatic cases should be performed to verify the
prognostic efficacy of determination of different
parameters in fetal blood.
At present, fetal blood sampling at 20-21 weeks gestation
cannot be justified for the prognosis of fetal CMV
infection due to the high risk of fetal demise.
Lazzarotto
Tiziana
Now
I would like to conclude my presentation
with the latest information on the treatment
of CMV-infected pregnant women
Lazzarotto
Tiziana
CHIP
-blind, placebo
-controlled, prospective
CHIP study:
study: aa randomized,
randomized, double
double-blind,
placebo-controlled,
prospective trial
trial
(phase
-specific hyperimmune
(phase 2B)
2B) for
for the
the evaluation
evaluation of
of the
the efficacy
efficacy of
of CMV
CMV-specific
hyperimmune globulin
globulin
Cytotect
® administered
Cytotect®
administered in
in pregnant
pregnant women
women with
with acute
acute phase
phase of
of primary
primary CMV
CMV
infection
infection for
for prevention
prevention of
of intrauterine
intrauterine transmission
transmission (Italian
(Italian study).
study).
Cytotect
® study:
Cytotect®
study: aa phase
phase 33 prospective
prospective trial
trial for
for the
the evaluation
evaluation of
of the
the efficacy
efficacy of
of CMV
CMV-specific
® administered
specific hyperimmune
hyperimmune globulin
globulin Cytotect
Cytotect®
administered in
in pregnant
pregnant women
women who
who
seroconverted
irus to
seroconverted in
in the
the first
first trimester
trimester to
to protect
protect against
against transmission
transmission of
of the
the vvirus
to the
the
unborn
unborn child
child and
and thereby
thereby prevent
prevent injury
injury to
to the
the child
child (European
(European study).
study).
Cymeval
-blind placebo
-controlled prospective
Cymeval study
study:: aa randomized
randomized double
double-blind
placebo-controlled
prospective trial
trial
evaluating
evaluating the
the efficacy
efficacy of
of valacyclovir
valacyclovir (the
(the oral
oral prodrug
prodrug of
of acyclovir).
acyclovir).
The
The drug
drug is
is given
given orally
orally to
to pregnant
pregnant women
women in
in cases
cases of
of confirmed
confirmed fetal
fetal CMV
CMV infection
infection
(amniotic
-positive) and
(amniotic fluid
fluid PCR
PCR-positive)
and with
with extracerebral
extracerebral signs
signs visible
visible on
on ultrasound
ultrasound that
that
can
can be
be attributed
attributed to
to the
the infection
infection for
for the
the reduction
reduction of
of number
number ooff cases
cases with
with
unfavourable
lly-indicated
unfavourable outcomes
outcomes (children
(children symptomatic
symptomatic at
at birth)
birth) and
and medica
medically-indicated
terminations
terminations of
of pregnancy
pregnancy (French
(French study).
study).
USA
USA -- Cytogam®
Cytogam® (CMV-IGIV)
(CMV-IGIV) study
study -- Eunice
Eunice Kennedy
Kennedy Shriver
Shriver National
National Institute
Institute of
of Child
Child
Health
Health and
and Human
Human Development
Development (NICHD):
(NICHD): aa randomized,
randomized, double-blind,
double-blind, placeboplacebocontrolled,
controlled, prospective
prospective trial
trial (phase
(phase 3)
3)
The
The purpose
purpose of
of this
this research
research study
study is
is to
to determine
determine whether
whether treating
treating pregnant
pregnant women
women
who
who have
have aa primary
primary CMV
CMV infection
infection with
with CMV
CMV antibodies
antibodies will
will reduce
reduce the
the number
number of
of
babies
babies infected
infected with
with CMV.
CMV.
Lazzarotto
Tiziana
CHIP
-blind, placebo
-controlled, prospective
CHIP study:
study: aa randomized,
randomized, double
double-blind,
placebo-controlled,
prospective trial
trial
(phase
-specific hyperimmune
(phase 2B)
2B) for
for the
the evaluation
evaluation of
of the
the efficacy
efficacy of
of CMV
CMV-specific
hyperimmune globulin
globulin
Published
study
in
NEJM
Cytotect
® administered
Cytotect®
administered in
in pregnant
pregnant women
women with
with acute
acute phase
phase of
of primary
primary CMV
CMV
April 3rd, 2014
infection
infection for
for prevention
prevention of
of intrauterine
intrauterine transmission
transmission (Italian
(Italian study).
study).
Cytotect
® study:
Cytotect®
study: aa phase
phase 33 prospective
prospective trial
trial for
for the
the evaluation
evaluation of
of the
the efficacy
efficacy of
of CMV
CMV-specific
® administered
specific hyperimmune
hyperimmune globulin
globulin Cytotect
Cytotect®
administered in
in pregnant
pregnant women
women who
who
This
was
an
open
single
arm
study.
seroconverted
irus to
seroconverted in
in the
the first
first trimester
trimester to
to protect
protect against
against transmission
transmission of
of the
the vvirus
to the
the
study
ended
this
year(European
unborn
thereby
prevent
injury
the
unborn child
child and
andThis
thereby
prevent
injury to
to
the child
child
(European study).
study).
and publication expected soon
Cymeval
-blind placebo
-controlled prospective
Cymeval study
study:: aa randomized
randomized double
double-blind
placebo-controlled
prospective trial
trial
evaluating
evaluating the
the efficacy
efficacy of
of valacyclovir
valacyclovir (the
(the oral
oral prodrug
prodrug of
of acyclovir).
acyclovir).
Estimated
Enrollment:
43
Pregnant
women
The
The drug
drug is
is given
given orally
orally to
to pregnant
pregnant women
women in
in cases
cases of
of confirmed
confirmed fetal
fetal CMV
CMV infection
infection
(amniotic
-Start
positive)
and
extracerebral
Date:
July
2011 signs
(amniotic fluid
fluid PCR
PCR-positive)
and with
with
extracerebral
signs visible
visible on
on ultrasound
ultrasound that
that
can
to
infection
for
can be
be attributed
attributedThis
to the
thestudy
infection
for the
the reduction
reduction
of number
number ooff cases
cases with
with
ended
in 2014of
unfavourable
lly-indicated
unfavourable outcomes
outcomes (children
(children symptomatic
symptomatic at
at birth)
birth) and
and medica
medically-indicated
and
publication
expected
soon.
terminations
terminations of
of pregnancy
pregnancy (French
(French study).
study).
USA
USA -- Cytogam®
Cytogam® (CMV-IGIV)
(CMV-IGIV) study
study -- Eunice
Eunice Kennedy
Kennedy Shriver
Shriver National
National Institute
Institute of
of Child
Child
Health
Health and
and Human
Human Development
Development (NICHD):
(NICHD): aa randomized,
randomized, double-blind,
double-blind, placeboplaceboEstimated
Enrollment:
800
Pregnant
women
controlled,
controlled, prospective
prospective trial
trial (phase
(phase 3)
3)
The
research
study
is
Date:
2012 whether
The purpose
purpose of
of this
thisStart
research
studyJuly
is to
to determine
determine
whether treating
treating pregnant
pregnant women
women
who
primary
CMV
who have
have aaCompletion
primary CMV
CMV infection
infection
with
CMV antibodies
antibodies
will reduce
reduce the
the number
number of
of
Date:with
December
2018will
babies
babies infected
infected with
with CMV.
CMV.
This study is currently IN PROGRESS.
Lazzarotto
Tiziana
Bando 2007
NCT00881517
C H I P study ⇒ Congenital HCMV Infection Prevention
Nigro
Nigro G.
G. et
et al.
al. NEJM
NEJM 2005
2005
Non
Non randomized
randomized study
study
Prevention
Prevention Group
Group
Received
Received HIG
HIG
Patients
Patients
hyper
-immune
hyper-immune
globulin
globulin
therapy/IV
therapy/IV
Congenital
Congenital CMV
CMV
37
37
47
47
100U
100U per
per kilogram
kilogram
every
every month
month until
until
delivery
delivery
16%
16%
40%
40%
P=0.04
P=0.04
Lazzarotto
Tiziana
NO
NO HIG
HIG
Revello
Revello MG
MG et
et al.
al. NEJM
NEJM 2014
2014
Randomized
Randomized study
study
Prevention
Prevention Group
Group
Received
Received HIG
HIG
NO
NO HIG
HIG
61
61
62
62
100
100 U
U per
per kilogram
kilogram
every
every month
month until
until
delivery
delivery
30%
30%
44%
44%
P=0.13
P=0.13
∆∆-14%
-14% 95%
-31.7/ +2.2)
95% CI
CI ((-31.7/
+2.2)
Current Issue: April 3, 2014
Lazzarotto
Tiziana
UNIVERSITY OF BOLOGNA
DIMES - School of Medicine
St. Orsola Malpighi University Hospital
Operative Unit of Clinical Microbiology
Laboratory of Virology
L. Gabrielli, G. Piccirilli, C. Pavia, A. Chiereghin
Department of Obstetrics and Gynecology
G. Simonazzi, B. Guerra, F. Cervi, N. Rizzo
Department of Pediatrics - Division of Neonatology
M. Lanari, M.G. Capretti, G. Faldella
Lazzarotto
Tiziana