Early Human Development 90S1 (2014) S29–S31
Diagnosis and counseling of fetal and neonatal HCMV infection Maurizio Zavattoni a, *, Milena Furione a , Alessia Arossa b , Angela Iasci b , Arsenio Spinillo b , Giuseppina Lombardi c , Mauro Stronati c , Andrea Righini d , Fausto Baldanti a a
Molecular Virology Unit, Microbiology and Virology Department, Fondazione IRCCS Policlinico San Matteo, Pavia, Italy Department of Obstetrics and Gynaecology, Fondazione IRCCS Policlinico San Matteo, Pavia, Italy Neonatology and Neonatal Intensive Care Unit, Fondazione IRCCS Policlinico San Matteo, Pavia, Italy d Radiology and Neuroradiology Department, Children’s Hospital V. Buzzi, Milano, Italy b c
A R T I C L E
I N F O
A B S T R A C T
Keywords: HCMV Congenital infection Prognostic markers
Fetal HCMV infection is investigated by amniocentesis when a maternal primary infection is diagnosed or ultrasound (US/MRI) abnormalities are observed. In fetal blood, prognostic markers of symptomatic congenital infection may be evaluated for parental counseling. At birth, viral load measurement in body fluids may correlate with long-term sequelae, but the prognostic accuracy of symptomatic infection increases when maternal, fetal, and neonatal parameters are combined. © 2014 Elsevier Ireland Ltd. All rights reserved.
1. Introduction
Furthermore, HCMV DNA load measurement in body fluids (urine and blood) at birth has been used to establish cut-off values in symptomatic and asymptomatic newborns, which may be correlated with long-term sequelae and used to monitor therapeutic interventions. This review summarizes current approaches in the diagnosis of congenital HCMV infection in the fetus and the newborn.
Human Cytomegalovirus (HCMV) can be transmitted from mother to fetus following either primary or recurrent infections. In case of primary maternal infection, in utero transmission rates increase from 30 to 40% around conception-first trimester up to 70% in the third trimester of pregnancy. At delivery, about 10– 15% congenitally infected newborns are symptomatic and 85–90% are asymptomatic. In utero HCMV infection acquired at an earlier gestational age seems to be associated with a worse outcome. On the contrary, the rate of congenital transmission from recurrent maternal infections is approximately 1.9%, and most cases are asymptomatic at birth. Permanent sequelae, such as sensorineural hearing loss (SNHL) and neurodevelopmental delay in congenitally infected symptomatic children are reported in 40–58% of cases, whereas in neonates without symptoms at birth, late permanent sequelae occur in 13.5%. Usually, fetal HCMV infection is investigated by amniocentesis when a maternal primary infection is diagnosed or ultrasound (US) abnormalities are observed. In Italy, therapeutic abortion is allowed up to 22 weeks’ gestation, thus in many cases the decision to terminate or to continue a pregnancy must be made in a narrow time frame. Therefore, it is of crucial importance that parental counseling be based upon accurate prognostic markers of symptomatic congenital infection at birth (or later in life). A combined assessment taking into account fetal blood examination, as well as, neuroimaging findings has been proposed to understand the events that lead to fetal damage and symptoms at birth.
* Corresponding author: Maurizio Zavattoni, Molecular Virology Unit, Microbiology and Virology Department, Fondazione IRCCS Policlinico San Matteo, Via Taramelli 5, 27100 Pavia, Italy. Tel.: +39 0382 502420; fax: +39 0382 502599. E-mail address: [email protected] (M. Zavattoni). 0378-3782/$ – see front matter © 2014 Elsevier Ireland Ltd. All rights reserved.
2. Discussion 2.1. Diagnosis of fetal infection HCMV fetal investigation is proposed when a primary HCMV infection is documented during pregnancy. Amniotic fluid (AF) is the sample of choice to investigate HCMV intrauterine transmission. HCMV can be detected in AF by rapid viral isolation (sensitivity, 81.8%) and quantitative PCR (sensitivity, 92.7%) [1]. However, delayed transmission cannot be ruled out in about 8% of cases, even when the best conditions for performing amniocentesis are respected (20 weeks’ gestation and at least 6–8 weeks from the onset of maternal infection). Fortunately, clinical outcome for these late-infected newborns is expected to be good. In case of a positive AF result, cordocentesis is recommended for prognostic purposes. Occasionally HCMV-related fetal US pathological findings are discovered in the absence of any serological screening for HCMV in pregnancy, or following indications when maternal primary HCMV infection has been diagnosed. Fetal morphological abnormalities include: (i) cerebral US features, i.e microcephaly, hydrocephaly, ventriculomegaly, increased periventricular echogenicity, calcifications, periventricular pseudocystis, intraventricular synechiae, malformations of cortical development, and cerebellar abnormalities; (ii) extracerebral (non-specific) abnormalities, i.e IUGR (intrauterine
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growth retardation), hydrops, ascites, pericardial effusion, pleural effusion, skin edema, hyperechogenic bowel, hepatomegaly/ splenomegaly; and (iii) placental abnormalities, i.e liver calcifications, placentomegaly, poly/oligo/anhydramnios. Cerebral US data collected at a mean gestational age of 25 weeks in HCMV-infected fetuses were compared with fetal MR imaging, and the latter showed a higher sensitivity in predicting symptomatic infection (83% vs 33%) [2]. Recently, the pattern of neurodevelopmental injury as a function of timing of acquisition of brain infection in utero was summarized by Lanari et al. [3]; the results suggest that gestational age at the time of maternal infection is an important factor in the risk for cerebral damage. 2.2. Diagnosis of neonatal infection Diagnosis of HCMV infection at birth relies on rapid virus isolation in tissue culture from urine in the first three weeks of life. Today, real-time PCR technology is preferred as it is more sensitive, standardized and less labour- and resource-intensive than tissue culture methods. The saliva PCR assay has been proposed for HCMV screening in newborns, since urine samples are more difficult to collect than saliva samples [4]. However, positive saliva results should be confirmed with PCR on urine due to possible maternal milk-contamination. In HCMV-infected newborns, it is recommended to collect blood samples for detection of antigenaemia, DNAemia, and IgM (sensitivity 46.0%, 98.6%, 54.0%, respectively). PCR testing of dried blood spots (DBS) (routinely obtained in all infants), has been proposed for the diagnosis of congenital HCMV infection. However, in a recent large-scale newborn screening study, this approach identified infants with congenital HCMV infection with a sensitivity of only 34.4% [5]. Thus, PCR DBS testing might be useful to retrospectively diagnose unexplained infections not investigated within the first three weeks of life. 2.3. Prognostic markers of symptomatic congenital infection 2.3.1. Gestational age at maternal infection The risk of vertical transmission and clinical outcome with reference to gestational age at maternal infection has been investigated by several groups [6–8]. Transmission varied between 8.3 and 16.7% in preconceptional infections; between 30.8 and 34.5% in periconceptional infections; between 30.1 and 42.2% in first trimester infections; between 38.2 and 44.1% in second trimester infections; and between 64.1 and 73.3% in third trimester infections. Thus, it is crucial for counseling to discriminate between preconceptional and maternal infection during pregnancy. Most importantly, the incidence of symptomatic congenital infection was found to be influenced by gestational age at the time of maternal infection. In our experience, preconceptional infections seem to be invariably subclinical, while in periconceptional infections 33.3% of infected neonates; in first trimester infection 25.5%; in second trimester infection 14.3%; and in third trimester infection 8% were symptomatic at birth. Thus, parents should be reassured in preconceptional and late infections during pregnancy, while prenatal diagnosis and counseling should be offered in the presence of earlier infections, which are associated with a higher risk of serious HCMV-associated pathologies. 2.3.2. Amniotic fluid and fetal blood Detection of viral DNA in AF is the gold-standard for documenting HCMV intrauterine transmission, and viral load levels in AF have been advocated to distinguish between symptomatic and asymptomatic fetuses. Although the difference reached statistical significance (9.8×105 HCMV DNA copies/mL in asymptomatic and 3.7×106 HCMV DNA copies/mL in symptomatic fetuses, P=0.030),
a great overlap between HCMV DNA values in the two groups was observed (9). As far as it is presently known, only very low viral DNA load levels in AF (≤103 HCMV DNA copies/mL) may predict a good prognosis. Since predictive cutoff values suffer from inter- and intralaboratory variations, expression of HCMV DNA load in UI/mL would be a step forward in standardization of molecular assays. Biochemical fetal blood parameters, such as thrombocytopenia (platelet count 80 IU/mL), direct bilirubin (>4 mg/dL), and β2-microglobulin levels (>5 mg/L) have been associated with a poor outcome. More recently, virological markers such as antigenaemia, viraemia, DNAemia, and HCMV-specific IgM antibody have been evaluated in FB, along with biochemical and haematological parameters to identify fetal cord blood prognostic markers of symptomatic congenital HCMV infection. For each fetal blood marker with a significant difference between asymptomatic and symptomatic fetuses, a cutoff with the highest sensitivity and specificity was selected and the relevant AUC was calculated. The results of this study showed that a combination of 3 out of 4 altered markers, i.e β2-microglobulin (≥11.5 mg/L), platelet count (≤50,000/μL), IgM ratio (>3.0) and DNAemia (>30,000 copies/mL blood) had a PPV of 100% and a NPV of 93.8% [9]. Since these findings were published, both β2-microglobulin, platelet count, IgM ratio and DNA load in FB have been investigated prospectively to validate an algorithm to predict an unfavourable fetal outcome. In parallel, MR imaging was utilized to predict neonatal outcome (Table 1). 2.3.3. Neonatal blood The prognostic value of HCMV DNA load and IgM levels in blood has been investigated by several groups [10,11]. Congenital symptomatic infections were consistently associated with a higher blood viral DNA load at birth. However, a great overlap of values between symptomatic and asymptomatic newborns makes parental counseling quite difficult. In fact, while
Diagnosis and counseling of fetal and neonatal HCMV infection Maurizio Zavattoni a, *, Milena Furione a , Alessia Arossa b , Angela Iasci b , Arsenio Spinillo b , Giuseppina Lombardi c , Mauro Stronati c , Andrea Righini d , Fausto Baldanti a a
Molecular Virology Unit, Microbiology and Virology Department, Fondazione IRCCS Policlinico San Matteo, Pavia, Italy Department of Obstetrics and Gynaecology, Fondazione IRCCS Policlinico San Matteo, Pavia, Italy Neonatology and Neonatal Intensive Care Unit, Fondazione IRCCS Policlinico San Matteo, Pavia, Italy d Radiology and Neuroradiology Department, Children’s Hospital V. Buzzi, Milano, Italy b c
A R T I C L E
I N F O
A B S T R A C T
Keywords: HCMV Congenital infection Prognostic markers
Fetal HCMV infection is investigated by amniocentesis when a maternal primary infection is diagnosed or ultrasound (US/MRI) abnormalities are observed. In fetal blood, prognostic markers of symptomatic congenital infection may be evaluated for parental counseling. At birth, viral load measurement in body fluids may correlate with long-term sequelae, but the prognostic accuracy of symptomatic infection increases when maternal, fetal, and neonatal parameters are combined. © 2014 Elsevier Ireland Ltd. All rights reserved.
1. Introduction
Furthermore, HCMV DNA load measurement in body fluids (urine and blood) at birth has been used to establish cut-off values in symptomatic and asymptomatic newborns, which may be correlated with long-term sequelae and used to monitor therapeutic interventions. This review summarizes current approaches in the diagnosis of congenital HCMV infection in the fetus and the newborn.
Human Cytomegalovirus (HCMV) can be transmitted from mother to fetus following either primary or recurrent infections. In case of primary maternal infection, in utero transmission rates increase from 30 to 40% around conception-first trimester up to 70% in the third trimester of pregnancy. At delivery, about 10– 15% congenitally infected newborns are symptomatic and 85–90% are asymptomatic. In utero HCMV infection acquired at an earlier gestational age seems to be associated with a worse outcome. On the contrary, the rate of congenital transmission from recurrent maternal infections is approximately 1.9%, and most cases are asymptomatic at birth. Permanent sequelae, such as sensorineural hearing loss (SNHL) and neurodevelopmental delay in congenitally infected symptomatic children are reported in 40–58% of cases, whereas in neonates without symptoms at birth, late permanent sequelae occur in 13.5%. Usually, fetal HCMV infection is investigated by amniocentesis when a maternal primary infection is diagnosed or ultrasound (US) abnormalities are observed. In Italy, therapeutic abortion is allowed up to 22 weeks’ gestation, thus in many cases the decision to terminate or to continue a pregnancy must be made in a narrow time frame. Therefore, it is of crucial importance that parental counseling be based upon accurate prognostic markers of symptomatic congenital infection at birth (or later in life). A combined assessment taking into account fetal blood examination, as well as, neuroimaging findings has been proposed to understand the events that lead to fetal damage and symptoms at birth.
* Corresponding author: Maurizio Zavattoni, Molecular Virology Unit, Microbiology and Virology Department, Fondazione IRCCS Policlinico San Matteo, Via Taramelli 5, 27100 Pavia, Italy. Tel.: +39 0382 502420; fax: +39 0382 502599. E-mail address: [email protected] (M. Zavattoni). 0378-3782/$ – see front matter © 2014 Elsevier Ireland Ltd. All rights reserved.
2. Discussion 2.1. Diagnosis of fetal infection HCMV fetal investigation is proposed when a primary HCMV infection is documented during pregnancy. Amniotic fluid (AF) is the sample of choice to investigate HCMV intrauterine transmission. HCMV can be detected in AF by rapid viral isolation (sensitivity, 81.8%) and quantitative PCR (sensitivity, 92.7%) [1]. However, delayed transmission cannot be ruled out in about 8% of cases, even when the best conditions for performing amniocentesis are respected (20 weeks’ gestation and at least 6–8 weeks from the onset of maternal infection). Fortunately, clinical outcome for these late-infected newborns is expected to be good. In case of a positive AF result, cordocentesis is recommended for prognostic purposes. Occasionally HCMV-related fetal US pathological findings are discovered in the absence of any serological screening for HCMV in pregnancy, or following indications when maternal primary HCMV infection has been diagnosed. Fetal morphological abnormalities include: (i) cerebral US features, i.e microcephaly, hydrocephaly, ventriculomegaly, increased periventricular echogenicity, calcifications, periventricular pseudocystis, intraventricular synechiae, malformations of cortical development, and cerebellar abnormalities; (ii) extracerebral (non-specific) abnormalities, i.e IUGR (intrauterine
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growth retardation), hydrops, ascites, pericardial effusion, pleural effusion, skin edema, hyperechogenic bowel, hepatomegaly/ splenomegaly; and (iii) placental abnormalities, i.e liver calcifications, placentomegaly, poly/oligo/anhydramnios. Cerebral US data collected at a mean gestational age of 25 weeks in HCMV-infected fetuses were compared with fetal MR imaging, and the latter showed a higher sensitivity in predicting symptomatic infection (83% vs 33%) [2]. Recently, the pattern of neurodevelopmental injury as a function of timing of acquisition of brain infection in utero was summarized by Lanari et al. [3]; the results suggest that gestational age at the time of maternal infection is an important factor in the risk for cerebral damage. 2.2. Diagnosis of neonatal infection Diagnosis of HCMV infection at birth relies on rapid virus isolation in tissue culture from urine in the first three weeks of life. Today, real-time PCR technology is preferred as it is more sensitive, standardized and less labour- and resource-intensive than tissue culture methods. The saliva PCR assay has been proposed for HCMV screening in newborns, since urine samples are more difficult to collect than saliva samples [4]. However, positive saliva results should be confirmed with PCR on urine due to possible maternal milk-contamination. In HCMV-infected newborns, it is recommended to collect blood samples for detection of antigenaemia, DNAemia, and IgM (sensitivity 46.0%, 98.6%, 54.0%, respectively). PCR testing of dried blood spots (DBS) (routinely obtained in all infants), has been proposed for the diagnosis of congenital HCMV infection. However, in a recent large-scale newborn screening study, this approach identified infants with congenital HCMV infection with a sensitivity of only 34.4% [5]. Thus, PCR DBS testing might be useful to retrospectively diagnose unexplained infections not investigated within the first three weeks of life. 2.3. Prognostic markers of symptomatic congenital infection 2.3.1. Gestational age at maternal infection The risk of vertical transmission and clinical outcome with reference to gestational age at maternal infection has been investigated by several groups [6–8]. Transmission varied between 8.3 and 16.7% in preconceptional infections; between 30.8 and 34.5% in periconceptional infections; between 30.1 and 42.2% in first trimester infections; between 38.2 and 44.1% in second trimester infections; and between 64.1 and 73.3% in third trimester infections. Thus, it is crucial for counseling to discriminate between preconceptional and maternal infection during pregnancy. Most importantly, the incidence of symptomatic congenital infection was found to be influenced by gestational age at the time of maternal infection. In our experience, preconceptional infections seem to be invariably subclinical, while in periconceptional infections 33.3% of infected neonates; in first trimester infection 25.5%; in second trimester infection 14.3%; and in third trimester infection 8% were symptomatic at birth. Thus, parents should be reassured in preconceptional and late infections during pregnancy, while prenatal diagnosis and counseling should be offered in the presence of earlier infections, which are associated with a higher risk of serious HCMV-associated pathologies. 2.3.2. Amniotic fluid and fetal blood Detection of viral DNA in AF is the gold-standard for documenting HCMV intrauterine transmission, and viral load levels in AF have been advocated to distinguish between symptomatic and asymptomatic fetuses. Although the difference reached statistical significance (9.8×105 HCMV DNA copies/mL in asymptomatic and 3.7×106 HCMV DNA copies/mL in symptomatic fetuses, P=0.030),
a great overlap between HCMV DNA values in the two groups was observed (9). As far as it is presently known, only very low viral DNA load levels in AF (≤103 HCMV DNA copies/mL) may predict a good prognosis. Since predictive cutoff values suffer from inter- and intralaboratory variations, expression of HCMV DNA load in UI/mL would be a step forward in standardization of molecular assays. Biochemical fetal blood parameters, such as thrombocytopenia (platelet count 80 IU/mL), direct bilirubin (>4 mg/dL), and β2-microglobulin levels (>5 mg/L) have been associated with a poor outcome. More recently, virological markers such as antigenaemia, viraemia, DNAemia, and HCMV-specific IgM antibody have been evaluated in FB, along with biochemical and haematological parameters to identify fetal cord blood prognostic markers of symptomatic congenital HCMV infection. For each fetal blood marker with a significant difference between asymptomatic and symptomatic fetuses, a cutoff with the highest sensitivity and specificity was selected and the relevant AUC was calculated. The results of this study showed that a combination of 3 out of 4 altered markers, i.e β2-microglobulin (≥11.5 mg/L), platelet count (≤50,000/μL), IgM ratio (>3.0) and DNAemia (>30,000 copies/mL blood) had a PPV of 100% and a NPV of 93.8% [9]. Since these findings were published, both β2-microglobulin, platelet count, IgM ratio and DNA load in FB have been investigated prospectively to validate an algorithm to predict an unfavourable fetal outcome. In parallel, MR imaging was utilized to predict neonatal outcome (Table 1). 2.3.3. Neonatal blood The prognostic value of HCMV DNA load and IgM levels in blood has been investigated by several groups [10,11]. Congenital symptomatic infections were consistently associated with a higher blood viral DNA load at birth. However, a great overlap of values between symptomatic and asymptomatic newborns makes parental counseling quite difficult. In fact, while
