Diagnosis of CMV Infection in Pregnancy

MP Landini (Italy)

1999 The International Herpes Management Forum, all rights reserved.

Congenital CMV infection

Incidence 0.52.2%
(mean 1%)

1015% symptomatic at birth

8090% asymptomatic at birth

5% with classic CID 5% with symptoms

1020% with subtle delayed sequelae (progressive hearing loss, mental deficiency, etc)

~ 3 out of 1000 newborns suffer from a congenital CMV infection

Congenital CMV infection

...but the rate of congenital CMV infection can be higher Author Country Year Population Infection infected (%) Women HIV + AIDS

Doyle et al. TexasUSA 1996

6.5 21

Incidence of congenital CMV infection in Italy

1200 newborns tested (19971998)

(rapid virus isolation from urine within the first week of life)

13 positives

1.1%

Incidence of congenital CMV infection in Italy

500000 newborns/year

1.1%
5500 CMV-infected newborns/year

CMV impact CMV

~1500 infants/year will suffer from

Congenital CMV infection

Exogenous infection (primary or secondary)
Every secretion

Endogenous infection

Pregnant women Transplacental route Fetus

Congenital CMV infection

Congenital CMV infection and maternal immunity
Seropositive women Women who seroconverted during pregnancy Congenital CMV infection 1.2% (22/1828) 12.9% (15/116)

Preconceptional maternal immunity is protective against congenital CMV infection decreasing risk of infection by 91%
Stagno 1997

Seropositivity for CMV among blood donors (Bologna, Italy)

100 90 80 70 60 50 1977 1985 1994 1997

Seropositivity (%)

Primary CMV infection in pregnant women

Symptoms: clinically inapparent persistent fever, myalgia, sore throat, cervical adenopathy, extreme fatigue
In most cases frequently uncommon

mononucleosis syndrome
Laboratory findings: atypical lymphocytosis

elevated hepatic transaminases negative heterophile antibody response

Diagnosis of primary CMV infection in pregnant woman

Specific laboratory diagnosis

February 1994 June 1999

Diagnostic follow-up of CMV infection in pregnancy

Bologna, Italy

Primary CMV infection in pregnant woman

1) Pre-pregnancy serological status for CMV is known Seropositive Seronegative
IgG+/IgMLong-term seropositive person Minimum risk

IgG-/IgMPre-tested at 2 and 4 months IgG-/IgMIgG+/IgMigG-/IgM+ igG+/IgM+ At risk

No follow up

No follow up

Advanced diagnosis

Primary CMV infection in pregnant woman

2) Pre-pregnancy serological status for CMV is unknown

Diagnosis of primary CMV infection in pregnant woman

IgM

Poor reliability of the CMV-IgM kits available (1997)

Assay Wampole Total True False SensitivitySpecificity Overall Positive Negative Negative Positive Negative agreement 64 41 35 21 33 35 40 23 24 0 4 7 40 91 83 34 60 59 100 73 69 60

Technogenetics 59 Sclavo 21

Bouty
Eurogenetics Sorin Behringwerke

21
53 52 30

19
37 39 27 41 38

39
37 39 40 31 40

2
16 13 3 24 10

41
11 10 31 5 13

31
77 80 46 89 74

95
70 75 93 56 80

57
73 77 66 71 77

M.A. Bioproducts 65 Bioch & Diagnos 48

All IgM-pregnant women were considered at risk

Advanced diagnosis

Pregnant women at risk of transmitting CMV referred to our reference centre in Bologna for advanced diagnosis

Advanced CMV diagnosis

IgM confirmation by Western blot Determination of the IgG avidity index Isolation of the virus from urine, saliva and blood

A confirmatory test for CMV-IgM

New immunoblot
1) Contains both structural and nonstructural proteins 2) Reactivity to vp 150 can be confirmed with recpUL32 3) Agrees with consensus of different ELISAs 4) Is easy to standardize 5) Is easy to interpret
Vp150 Vp82 Vp65 Vp28 rp150 rp52 rp130 rp38 Recombinant proteins Purified native viral proteins

CKS

Congenital CMV infections

Low IgG avidity is linked to primary infection
70 60

Avidity index (%)

50 40 30 20 10 0 0 5 10 15 20 25 30 35

Weeks after beginning of symptoms

(I) Uninfected women*

IgG negative/IgM negative IgG positive/IgM negative (high avidity)

*Virus isolation negative

(II) Primary infections*

IgG negative/IgM positive IgG positive/IgM positive (low avidity)

*Virus isolation positive or negative

(III) Recurrent infections*

IgG positive/IgM positive (high avidity)

*Virus isolation positive or negative

(IV) Undefined infections*

Undefined presence of IgM Borderline avidity

*Virus isolation negative

19941999 Bologna, Italy

611 pregnant women at risk of transmitting CMV

Advanced diagnosis
4 Groups I Uninfected n=300 (49.1%) No congenital infections II Primary infections n=161 (26.3%) III Recurrent infections n=125 (20.5%) IV Undefined infections n=25 (4.1%) No congenital infections

YES No congenital congenital infections infections n=39 (24.4) With symptoms No symptoms n=18 (46.2%) n=21 (53.8%)

Main diagnostic problem

Approximately 20% of primarily infected mothers will give birth to an infected newborn

How to discriminate high-risk from low-risk pregnant women?

Prenatal diagnosis of congenital CMV infection

Prevent unnecessary termination in 70% of cases Give the possibility of termination in 30% of cases

Why?

The newborn will be checked for CMV infection and, if necessary, treated with ganciclovir Prenatal therapy (?)

Transmission of CMV through the placenta barrier and infection of the fetus
Infected mother viraemia infection of placenta trophoblasts

Infection of the oropharynx Virus in amniotic fluid Infection of fetal endothelial cells

Fetal viruria Viral replication in target organs (kidney)

Fetal viraemia

Prenatal diagnosis of congenital infection

1) Because some false-negative results have been reported before this age as the fetus excretes CMV via urine into the amniotic fluid and fetal diuresis only becomes established after 2021 weeks gestation 2) Because in most cases at least 69 weeks have to pass from the time of maternal infection before virus can be detected in amniotic fluid and it is known that CMV transmission is correlated with severe fetal diseases mainly when occurs during the first 12 weeks gestation

When?
2123 weeks gestation

Prenatal diagnosis was offered to all women undergoing a primary CMV infection and to those with an undefined type of infection

(I) 300 uninfected women

280 stop the follow-up (93.3%)
253 newborns checked Congenital infections=0
SV=shell vial PCR=polymerase chain reaction

20 requested amniocentesis
SV-positive=0 PCR-positive=0 Congenital infections=0

(III) 125 recurrent infections

115 (92%) stop the follow-up 10 requested amniocentesis
SV-positive=0 PCR-positive=1 Congenital infections=0

94 newborns checked Congenital infections=0

SV=shell vial PCR=polymerase chain reaction

(IV) 25 undefined type of infection

11 did not accept amniocentesis
7 newborns checked Congenital infections=0
SV=shell vial PCR=polymerase chain reaction

14 requested amniocentesis
SV-positive=0 PCR-positive=2 Congenital infections=0

(II) 161 primary infections

82 (50.9%) did not accept amniocentesis
65 newborns were checked Congenital infections=17 (26.1%)
SV=shell vial

79 (49.1%) accepted amniocentesis

SV-positive=16 PCR-positive=42 Congenital infections=22 (27.8%)

PCR=polymerase chain reaction

Shell vial in the amniotic fluid and congenital infection

Congenital infection Yes No Total
Shell vial

16

16

6
22

57
57

63
79

Sensitivity: 72.7% Positive predictive value: 100%

Specificity: 100% Negative predictive value: 90.5%

PCR in amniotic fluid and congenital CMV infection

Congenital infection Yes No Total
PCR

22

20

42

0
22

37
37

37
79

Sensitivity: 100% Positive predictive value: 52.4%

Specificity: 64.9% Negative predictive value: 100%

qPCR in amniotic fluid and congenital CMV infection

Cutting point 103 GE/ml amniotic fluid Congenital infection Yes No Total
qPCR

103 <103

17 5 22

0 1 1

17 6 3

Sensitivity: 77.3% Positive predictive value: 100%

Specificity: 100% Negative predictive value: 82.8%

Prenatal diagnosis can identify congenital infections prenatally Can prenatal diagnosis identify fetuses at higher risk of CMV-related symptoms?

161 primary infections

144 newborns/fetuses checked
39 congenital infections (27.1 %)

31 newborns
13 symptomatic (41.9%) 18 asymptomatic (58.0%)

8 fetuses
5 disseminated infections + macroscopic evidences of damage 3 disseminated infections

39 congenital infections
22 mothers had accepted amniocentesis
14 newborns 7 symptomatic 7 asymptomatic 8 fetuses 5 disseminated infections + macroscopic evidences of damage 3 disseminated infections

Results of prenatal diagnosis performed on primarily infected mothers in relationship with pregnancy outcomes
Case Ultrasonographic findings# 1 Normal 2 3 4 5 6 Normal Central ventriculomegaly Prenatal diagnosis on amniotic fluid Shell PCR qPCR6 + + >1.8x10 (GE/ml) + + >1.8x106 + + 1.1x106 + + + + + + 9.1x105 9x105 5.5x105 Outcome ToP/disseminated CMV infection (diffused microcalcified cerebral areas, hepatosplenomegaly, pneumonitis) CMV infected newborn with mild hepatitis, high CRP ToP/disseminated CMV infection, pneumonitis, agensis of corpus callosum, hepatomology ToP/disseminated CMV infection, hepatosplenomegaly, pneumonitis CMV-infected newborn with cerebral vasculitis ToP/disseminated CMV infection, Dandy Walker syndrome, borderline cerebral ventriculomegaly CMV-infected newborn, small for gestational age* Fetus with borderline cerebral ventriculomegaly resolved in utero. Newborn with mild hepatitis CMV-infected newborn with severe cerebral ventriculomegaly, diffused microcalcified cerebral areas, hepatosplenomegaly** CMV-infected newborn with depression of the platelets and cerebral microcalcification ToP/borderline cerebral ventriculomegaly and CMV-infections in liver and lung CMV-infected newborn with mild hepatitis ToP/disseminated CMV infection ToP/disseminated CMV infection ToP/disseminated CMV infection

Normal Normal Vermis defects, borderline cerebral ventriculomegaly hyperchogenic bowel 7 Normal 8 Borderline cerebral Ventriculomegaly 9 Cerebral ventriculomegaly hyperchogenic bowel 10 Normal 11 Borderline cerebral ventriculomegaly, hyperchogenic bowel 12 Normal 13 Normal 14 Normal 15 Normal
ToP: termination of pregnancy # detected at 2022 weeks gestation

+ + + + + + +

+ + + + +

4x105 3.7x105 2.9x105 1.7x105 5x104

+ 5.0x103 + 21.8x106 + 3.1x105 + 1.7x105

*psychomotor reduction after first year **chrioretinitis and mental retardation (IQ<70) after first year

qPCR in 79 amniotic fluids relationship with pregnancy outcome

N= newborns F= fetuses F N F N N N F N N F F N

F
F F

CMV-GE/ml

106 105 104 103

F N (n 56)
Uninfected

NN N NN
Infected asymptomatic Infected symptomatic

Pregnancy outcomes

Main conclusions
The screening for CMV in pregnancy carried out by IgM and IgG avidity identifies all women who will deliver an infected infant The determination of the viral load in the amniotic fluid by qPCR can:
identify a congenital infection prenatally identify fetuses prenatally at higher risk of developing CMV-related symptoms (work still at the investigational stage)

Factors that may be involved in determining the severity of congenital CMV infection
Maternal viral load The strain of CMV The repertoire of CMV-specific maternal antibodies Factors linked to a good or weak placenta barrier

The viral load in the fetus

Co-infection with other viruses

University of Bologna Medical School

Department of Clinical and Experimental Medicine Section of Microbiology and Virology

MP Landini T Lazzarotto S Varani P Spezzacatena L Gabrielli Department of Obstetrics and Gynecology B Guerra Department of Pediatrics Division of Neonatology M Lanari

Groups of pregnant women

Congenital CMV infection (%)

Symptomatic congenital CMV infection (%)

(newborns + fetuses with macroscopic damages)

IgM-positive

14.2

6.6

With low anti-CMV IgG avidity With PCR+ amniotic fluid With 103 GE/ml of amniotic fluid

26.2

12.1

50

29.3

100

85.7

With 105 GE/ml of amniotic fluid

100

100

Congenital CMV infection diagnostic follow-up

IgG/IgM automated test +/ latently infected /+ +/+ / Second test 16-18 weeks gestation / No infection Third test 34-37 weeks gestation / Within the 18 weeks gestation
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Laboratorio di Virologia Molecolare Centro di Diagnosi della infezione da Citomegalovirus - Bo

First test

FIRST LEVEL

/+ +/ +/+
Avidity test -IgG Immunoblot-IgM latently recurrent

SECOND LEVEL

/+ +/ +/+ Neo-natal and post-neonatal follow-up

primary

OBSTETRIC COUNSELLING

PRENATAL DIAGNOSIS

21-23 weeks gestation

Congenital CMV infection diagnostic follow-up

PRENATAL DIAGNOSIS
Amniocentesis ultrasonographic examination Amniotic fluid PCR and/or virus isolation

Laboratorio di Virologia Molecolare Centro di Diagnosi della infezione da Citomegalovirus - Bo

+
qPCR

OBSTETRIC COUNSELLING
AUTOPTIC EXAMINATION FOR THE ABORTED FETUSES

LOW RISK NO RISK

HIGH RISK First week of life

Virus isolation from urine Antigenemia - Viraemia

EXAMINATIONS FOR THE NEWBORN

Virus isolation from urine

+
NEO-NATAL FOLLOW UP

Second week of life

NEO-NATAL FOLLOW UP

Newborns with congenital infection

+
START FOLLOW-UP POST-NEONATAL

Third month of life

Sensitivity, specificity, positive predictive value and negative predictive value of virus isolation and PCR in the presence or absence of CMV in the fetus/newborn
CMV infection Yes No SNS* SPE* PPV* NPV* Virus isolation Positive Negative PCR Positive 16 6 22 0 72.7 57 20 100 64.9 52.4 100 100 100 90.5

Negative

37

*: SNS (number of true positives/total number of infection) x 100 SPE (number of true negatives/total number of np infection) x 100 PPV (number of true positives/number of true positives + number of false positives) x 100 NPV (number of true negatives/number of true negatives + number of false negatives) x 100

Antibody affinity: the strength of a single antigenantibody bond

high-affinity high attraction Good fit low-affinity low attraction Poor fit

Antibody avidity: the strength with which a multivalent antibody binds a multivalent antigen