Abstract. Congenital cytomegalovirus (CMV) infection isthe leading cause of mental retardation and hearingimpairment. Examination for the presence of CMV infectionwas carried out in a selected population of 70 neonates.Urine samples were tested for CMV by means of a nestedpolymerase chain reaction. CMV was detected in 6 (16.7%)of the 36 preterm newborns and in 5 (14.7%) of the 34 full-term newborns. One preterm neonate died and the remaining10 newborns were followed up. Two children born at full-termdid not excrete CMV at 2 years of age and were symptom-free. Of 8 CMV-excreting children (5 preterm and 3 full-term), 2 were symptom-free (1 preterm and 1 term).Symptomatic CMV disease developed in 6 children (4preterm and 2 full-term), with mental retardation (n=4),hearing loss (n=1), strabismus (n=2) or bronchial asthma(n=1). Screening of such neonates is important; thoseidentified as congenitally CMV-infected can be monitored tocorrect any sequelae immediately.

Human cytomegalovirus (CMV) is one of the eight

members of the Herpesviridae family. It is a double-

stranded DNA virus that is restricted to humans. Epithelial

cells, endothelial cells and fibroblasts are the major targets

of CMV infection in vivo (1). In some instances, neurones

in the brain and retina, smooth muscle cells in the

gastrointestinal tract and hepatocytes can also be infected.

In the peripheral blood, the circulating leukocytes are

susceptible to CMV. In vitro, the virus replicates well only

in human fibroblasts. CMV displays many of the

characteristics of the herpesvirus family, such as the ability

to cause primary infection, then evolve into a latent state

and subsequently undergo reactivation.

CMV is the most common pathogen of intrauterine

infections, affecting an estimated 1-2% of all live births.

However, the incidence of congenital infection among

different populations is quite variable (2). Maternal primary

CMV infection occurs in approximately 0.7% to 4% and is

usually asymptomatic. Subsequent vertical transmission to

the foetus has been demonstrated at rates of 24% to 75%

(2-4). The usual clinical manifestations of congenital

infection are intrauterine growth retardation (IUGR),

microcephaly, ventriculomegaly, intracranial calcification,

chorioretinitis, hepatosplenomegaly, jaundice, petechiae,

thrombocytopenia and elevated serum transaminase levels

(5). The seriously ill babies have a mortality rate of

approximately 30% (6,7). The most common long-term

sequelae in infancy and childhood are sensorineural hearing

deficits and learning disabilities. Many infants, who appear

normal at birth, will manifest a hearing loss and other

delayed neurological complications. A progressive

sensorineural hearing loss may develop in 13-15% of

asymptomatic newborns (8).

A substantial proportion of congenital CMV infections

occur in women with preconceptional immunity (9-11). A

majority of infants with congenital CMV, who are born to

women with preconceptional immunity acquire the virus as

a result of recurrent maternal infection: reactivation of

endogenous virus or reinfection with a new strain of CMV.

A number of studies have suggested that symptoms and

permanent neurological deficits are rare in the infants of

women with preconceptional immunity (3,4). However, such

a clinical outcome may, in fact, be more common than

previously thought (12-15).

In the present study, the incidence, clinical spectrum and

outcome of congenital CMV infection were investigated

819

Correspondence to: Rozália Pusztai, Department of Medical

Microbiology and Immunobiology, University of Szeged, H-6720

Szeged, Dóm tér 10, Hungary. Tel: 36-62-546112, Fax: 36-62-

545113, e-mail: pusztai@comser.szote.u-szeged.hu

Key Words: Cytomegalovirus, congenital infection, preterm

neonates, full-term neonates, intensive care.

in vivo 18: 819-824 (2004)

Incidence and Outcome of Congenital CytomegalovirusInfection in Selected Groups of Preterm and Full-term

Neonates Under Intensive CareARANKA NAGY1, EMÖKE ENDREFFY1, KÁROLY STREITMAN1,

SÁNDOR PINTÉR1 and ROZÁLIA PUSZTAI2

1Department of Paediatrics and 2Department of Medical Microbiology and Immunobiology, Faculty of Medicine, Albert Szent-Györgyi Medical and Pharmaceutical Centre, University of Szeged, Hungary

0258-851X/2004 $2.00+.40

among preterm and full-term newborns under intensive care

with suspected intrauterine infection. Paediatric, otologic,

ophthalmologic and neurological follow-ups were scheduled

for 1 to 4 years.

Materials and Methods

Patients. Seventy newborns aged less than 2 weeks were admitted

to the Intensive Care Unit of the Department of Paediatrics,

University of Szeged, Hungary, with suspected intrauterine

infection (gestational age 25-40 weeks, weight 750-4500 g). Virus

infection was suspected in consequence of the detection of an

abnormality such as IUGR, non-immune hydrops foetalis (NIHF),

ascites, hydrothorax, congenital malformation, sepsis, hyper-

bilirubinaemia, seizure or pneumonitis. Clinical information was

obtained by review of the medical records of each subject. All

neonates with proven congenital CMV infection were followed-up

by the same paediatrician, ocular specialist, otologist and

neurologist. They were examined 2 weeks after birth and once or

twice annually up to 1-4 years of age.

The control group comprised 50 infants at no risk of foetal

CMV transmission.

The present study was approved by the Human Investigation

Review Board of the University of Szeged.

Detection of CMV in urine. Urine samples were collected from the

neonates in sterile urine-collecting bags within the first 2 weeks of

life, aliquoted into Eppendorf tubes and stored at -70ÆC until

processed. CMV DNA was extracted from 200 Ìl of the urine

sample by using the High Pure Viral Nucleic Acid Kit (Roche

Diagnostics GmbH, Mannheim, Germany) according to the

manufacturer’s instructions. Briefly, cell and virus lysis was

performed by incubation of the samples in a lysis/binding buffer

with proteinase K, and poly(A) carrier RNA and CMV DNA were

selectively bound to a glass fleece by centrifugation. Residual

impurities were removed in washing steps and the bound crude

CMV DNA was eluted in 50 Ìl of nuclease-free sterile water. The

viral DNA target sequences derived from the envelope

glycoprotein B (gB) were amplified by nested polymerase chain

reaction (nPCR). The outer primer pair consisted of

oligonucleotide 5’-GGA AAC GTG TCC GTC TTT GA-3’ (gB

1246) and 5’-GAG TAG CAG CGT CCT GGC GA-3’ (gB 1724).

The inner primer pair consisted of the sense primer 5’-TGG AAC

TGG AAC GTT TGG C-3’ (gB 1319) and the antisense primer 5’-

GAA ACG CGC GGC AAT CGG-3’ (gB 1604).

Amplification was carried out in a final volume of 50 Ìl

containing 50 pmol of primers, 200 ÌM dNTPs (each), 1.5 mM

MgCl2 and 1.25 Units of Taq DNA polymerase in 50 mM KCl, 10 mM

Tris-HCl (pH 9.0), 0.1% Triton X-100 reaction buffer. Five Ìl

aliquots of the crude DNA preparations were added to the first

PCR, and 3 ml aliquots from the previous PCR to the second one.

The thermal cycling profile was as follows: denaturation for 5 min

at 95ÆC, 35 cycles of amplification (95ÆC for 30 sec, 55ÆC for 40

sec and 72ÆC for 60 sec) and then holding at 72ÆC for 10 min. A

positive control with DNA isolated from the Towne strain of CMV

was included in each experiment. In both the initial and nPCR

reactions, negative controls were also included in each experiment

(buffer, nucleotides, primers, magnesium and Taq DNA

polymerase, but no template). PCR products (approximately 285 bp)

were detected by electrophoresis in 2% agarose gel stained with

ethidium bromide.

As the primers for amplification were selected from a region of

high peptide variability in the gB gene, the gB genotype of CMV

DNA was also determined by restriction fragment length

polymorphism (16).

Results

Seventy ill neonates were enrolled in the CMV follow-up

study: 36 preterm newborn infants and 34 term newborn

infants.

The presence of CMV DNA in the urine of the neonates

was detected by means of nPCR, and the gB genotype of the

CMV was also determined. CMV was found in the urine

samples of 11 (15.7%) of the neonates under intensive care.

Of the 11 CMV DNA samples, 10 proved to be of gB1 and

1 of gB2 genotype (case 9). During the follow-up period,

specimens for CMV detection were available from 10

patients. The gB genotype of the CMV DNA was found to

be the same as indentified after birth. Urinary excretion of

CMV was not detected in any of the 50 healthy neonates.

In 4 of the CMV-infected neonates, hypoxaemia was

detected at birth. In these cases, the Apgar scores were less

than 4 at 1 minute (Table I). The pH of the arterial blood was

below 7.2 in 3 cases. The paO2 was below 50 mm Hg in 3

cases, but the O2 therapy applied resulted in a saturation

efficiency of 89-95% (Table I). In 2 cases, hyperbilirubinaemia

was detected, and in 2 cases also thrombocytopenia (Table II).

Following clinical admission, 7 neonates, of whom 3 were

preterm (cases 1, 2 and 4) and 4 were full-term (cases 3, 5,

6 and 9), required ventilatory support for 1 to 10 days.

Patient 4, a preterm infant, died of intracranial

haemorrhage, septic uraemia and prematurity at 9 days of

age. Ten CMV-infected neonates were followed up for 5

in vivo 18: 819-824 (2004)

820

Table I. Clinical features in 11 newborn infants with congenital CMVinfection.

Infant Apgar scores at Arterial blood

1, 5 and 10 min pH PaO2 (mm Hg) O2SAT (%)

1 6/5/5 7.17 43.7 91

2 5/7/9 7.20 50.0 92

3 0/1/2 7.10 52.6 80

4 2/5/5 7.23 45.0 90

5 6/7/8 7.20 55.0 91

6 2/3/4 7.12 62.3 90

7 1/4/7 7.30 57.0 93

8 9/9/10 7.32 80.0 95

9 8/6/7 7.22 43.7 85

10 9/10/10 7.52 79.0 96

11 7/8/9 7.26 55.2 70

months to 4 years (Table III). Mental retardation was

observed in 4 cases, of whom 2 were preterm (cases 1 and 2)

and 2 were full-term neonates (cases 3 and 5). A

sensorineural hearing loss was detected in 1 neonate (case 3),

who was also growth-retarded. Bronchial asthma developed

in 1 preterm (case 10) and strabismus in 2 preterm cases

(cases 1 and 7). Five children born preterm (cases 1, 2, 7, 10

and 11) excreted CMV at their last visit and 1 was symptom-

free. In the full-term group, 3 of the 5 patients excreted virus

and 1 was symptom-free. No virus was detected in the urine

of the 2 full-term patients (cases 6 and 9) during the follow

up period. They were clinically symptom-free, but intense

mechanical ventilation was required at birth in both cases.

Ophthalmologic examination did not reveal chorioretinitis in

any of the cases. Strabismus developed in 2 preterm infants

(cases 1 and 7). The results of audiometric testing were found

to be normal in all infants except case 3.

Discussion

The prevalence of congenital CMV infection in selected

newborns under intensive care with suspected intrauterine

infection proved to be high (15.7%). The frequency of CMV

infection in the preterm newborns (16.7%) was similar to

that observed in the full-term newborns (14.7%). Santos etal. (17) detected congenital CMV infection in 6.8% (20 out

of 292) of non-selected newborns in a neonatal intensive

care unit. In Brazil, where the rate of CMV seropositivity

among pregnant women is 95%, Yamamoto et al. (18)

observed that the frequency of congenital CMV infection in

non-selected preterm newborns (2.1%) was similar to that

in full-term newborn infants (1.8%) and to that in another

high-immunity population (19,20).

The occurrence of severe manifestations of congenital

CMV infection has been reported in preterm infants

(19,21). In our study, 1 of the 6 preterm infants (case 4),

who was born with intracranial haemorrhage, sepsis and

RDS, died at 9 days of age. Another preterm infant (case 2)

was similarly born with intracranial haemorrhage. This

infant had pneumonitis at birth, too. An association

between congenital CMV infection and intracranial

haemorrhage has been described in two previous reports

(22,23). As 1-2% of babies are born with CMV infection, it

may be important to test for CMV in infants with

intracranial haemorrhage where there is no immediately

apparent cause. Necrotizing enterocolitis (NEC) was the

only clinical finding in 1 preterm infant (case 1) at birth, and

at 12 months of age mental retardation and strabismus were

detected. The association of NEC with congenital CMV

infection is rare. During the past 20 years, 6 cases of CMV-

associated NEC have been reported in infants. The last

case, in a premature infant born to a mother with recurrent

maternal CMV infection, was published recently (24). Our

CMV-associated NEC case confirms the suggestion of

others that CMV should be added to the list of pathogens

potentially responsible for NEC (24,25). However, further

studies are needed, as full-term neonates with congenital

CMV infection-associated NEC have not been identified

(26). Strabismus occurs more frequently among children

who are symptomatic at birth than among those who are

initially asymptomatic (27,28). Although additional evidence

is needed, our data suggest that the incidence of strabismus

in the preterm population may be higher than in the full-

term population. The prolonged urinary excretion of CMV

was documented in both asymptomatic and symptomatic

patients in previous studies (29-31).

Nagy et al: Congenital CMV Infection in Selected Groups of Neonates

821

Table II. Laboratory findings in 11 newborn infants with congenital CMV infection at 2 weeks of age.

Infant Thrombocyte Blood serum

number (G/l) Bilirubin GOT GPT CN Creatine

(ÌM) (U/l) (U/l) (mM) (ÌM)

1 240 154.4 22 17 10.9 119

2 204 87.8 22 16 6.6 55

3 191 42.1 NT NT 6.7 162

4 128 133.6 45 57 16.6 207

5 56 NT* NT NT 9.9 139

6 237 27.1 NT NT 7.8 45

7 103 236 54 17 4.4 77

8 199 239 18 23 6.0 118

9 268 NT 15 13 4.9 120

10 132 102 25 19 4.8 98

11 238 186 24 20 1.8 89

* NT: not tested

GOT: glutamate-oxalate-transaminase; GPT: glutamate-pyruvate-transaminase; CN: carbamic nitrogen

In conclusion, our study has demonstrated that the

frequency of congenital CMV infection is high in preterm

(16.7%) and full-term (14.7%) newborn infants under

intensive care with suspected intrauterine infection. It seems

that the clinical findings and the gB genotype of CMV

detected at birth do not allow a prediction of the outcome

of congenital CMV infections. Intracranial haemorrhage and

NEC should be added to the possible sequelae of congenital

CMV infection, especially in preterm newborns. Screening

of this group of neonates for congenital CMV appears

important. Those identified as having congenital CMV

infection can be monitored to correct any sequelae

immediately. The compassionate use of ganciclovir in

newborns with life-threatening or vision-threatening

congenital CMV infection is probably justified (32).

Acknowledgements

This study was supported by the Hungarian Scientific Research

Fund (grant OTKA-T26442/1998), the Research Fund of the

Hungarian Ministry of Education (grant FKFP 113/2000), and

ICON-H3 Ltd.

We thank Mrs. Ildikó Wellinger and Mrs. Csilla Szabó for their

excellent technical assistance.

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in vivo 18: 819-824 (2004)

822

Table III. Clinical findings in neonatal period and outcome of congenital CMV infection in newborns under intensive care.

Infant Gestation Body weight Clinical findings CMV excretion

at (g) in neonatal period and up to 48 months (months)*

age

(weeks)

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(12)

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Received July 30, 2004Accepted October 20, 2004

Nagy et al: Congenital CMV Infection in Selected Groups of Neonates

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