Fetal and Pediatric Pathology, 33:64–70, 2014 C Informa Healthcare USA, Inc. Copyright ISSN: 1551-3815 print / 1551-3823 online DOI: 10.3109/15513815.2013.856500
ORIGINAL ARTICLE
Fetal Pediatr Pathol Downloaded from informahealthcare.com by University of Newcastle on 09/30/14 For personal use only.
Diagnosis of Congenital Cytomegalovirus Antigenemia by Immunohistochemical Detection of Immediate Early Antigen A. Seval Ozgu-Erdinc1 and Cihat Sen2 1 Zekai Tahir Burak Women Health Care, Education and Research Hospital, Perinatology, Ankara, Turkey; 2 Istanbul University Cerrahpasa School of Medicine, Division of Maternal Fetal Medicine, Department of Obstetrics and Gynecology, Istanbul, Turkey
Cytomegalovirus (CMV) can be a cause of fetal morbidity and mortality among approximately 1% of pregnancies. With an aim to detect CMV antigenemia among 51 pregnant women with/without clinically diagnosed abnormalities and intrauterine growth retardation (IUGR) maternal and fetal samples either prenatal (n:22) or postpartum (n:29) were obtained between 17–42 weeks of gestation to analyze anti-CMV IgG, IgM antibodies and cytoplasmic or nuclear CMV antigens. Cytoplasmic and nuclear CMV antigenemia was detected among 19.6% and 11.8% of maternal samples. These values were 29.4% and 17.6% for fetal samples. Among both maternal and fetal samples, there was a 100% correlation when IgG and IgM were negative. The correlation for IgG and IgM positivity was not present among maternal samples since cytoplasmic (37.5%) and nuclear (25%) antigens could not be demonstrated in spite of immunity. Cytoplasmic and nuclear CMV antigens were detected within fetal samples from subjects presenting maternal immunoglobulin positivity, clinical abnormality and clinically normal findings (50, 32, 16.7% and 50, 16, 5.6%) respectively. In conclusion, immunocytochemical detection of CMV antigenemia improves CMV infection diagnosis which may be associated with clinical abnormalities/IUGR. Keywords: CMV, antigenemia, diagnosis, congenital CMV
INTRODUCTION Cytomegalovirus (CMV) is the most common cause of congenital infection affecting 0.2% to 2.3% of births worldwide. Furthermore it is the leading infectious cause of sensorineural hearing loss and mental retardation [1, 2]. High rates of congenital CMV infections have been consistently found in populations with a high seroprevalence [3]. Congenital CMV infection rates increase in parallel with maternal seroprevalence due to transmission from maternal nonprimary infection or reactivation [4]. While CMV IgG seropositivity have been detected among 94–99% of healthy subjects CMV IgM have been found to be positive among 0.2–1.4% of pregnant women in Turkey [5–9]. Although it does not correlate with the risk of antenatal infection, the rates of virus excretion via urine and cervical secretion of pregnant women range from 3 to 12% and
Received 19 July 2013; Revised 11 October 2013; accepted 14 October 2013. Address correspondence to Dr A. Seval Ozgu-Erdinc, Zekai Tahir Burak Women Health Care, Education and Research Hospital, Perinatology, Angora caddesi Ergonul Sitesi No:13 Cayyolu, Ankara, 06810 Turkey. E-mail: [email protected]
Fetal Pediatr Pathol Downloaded from informahealthcare.com by University of Newcastle on 09/30/14 For personal use only.
Diagnosis of Congenital Cytomegalovirus Antigenemia
from 11 to 28%, respectively [10]. A study from Canyılmaz et al. showed that cervical CMV excretion rate during the third trimester of pregnancy was 10.75% [10]. Fetal transmission and teratogenicity at birth or in utero is relatively rare, (10%) of CMV antigens in maternal urine. The risk of intrauterine infection largely depends on the time of maternal infection during pregnancy. Primary CMV infection occurs during pregnancy in 2% of women, with intrauterine transmission in approximately 40% of the cases [11]. Approximately 10% of the infected live born neonates have symptomatic disease at birth. In addition, between 7 and 15% of the asymptomatic newborns develop late squeal, particularly sensorineural hearing loss and neurodevelopmental disorders [12]. Most cases are subclinical in early life and some may present as sensorineural hearing loss when starting at school or during screening tests. The diagnosis of congenital infection can be made by demonstration of CMV; by isolation of virus from urine (Culture techniques); by detection of CMV-IgM in blood (Serological tests); detection of CMV antigen (Antigenemia assay) in blood samples and identification of CMV-DNA by PCR from samples [12–15]. Over the past several years, the direct detection of pp65 antigen in blood leukocytes has been successfully applied to the early diagnosis of CMV infections [16, 17]. As the method permits an early diagnosis before the onset of clinical symptoms, either termination of early gestational week pregnancies or targeted preventive treatment using hyper immunoglobulin is possible. Thus due to its frequent observation and potential complications detection of CMV is needed. With an aim to determine the frequency of CMV antigenemia in our center among pregnant subjects and umbilical cord blood we analyzed the presence of CMV by serological and immunohistochemical methods at a time period when PCR technology for detection of CMV DNA was not available. METHODS This cross-sectional study was conducted in the Istanbul University Cerrahpasa School of Medicine, department of obstetrics and gynecology, division of maternal fetal medicine, Istanbul, Turkey. A total of 51 pregnant women and their offsprings’ (22 fetuses and 29 newborn) were included in the study as shown in Table 1. Blood samples were obtained from 25 pregnant subjects with fetal abnormalities suggesting CMV infection (oligohydramnios, polyhydramnios, intrauterine growth retardation (IUGR), echodensities of bowel, liver or ventricle cerebral ventriculomegaly or atrophy, hydrocephalus, microcephaly, cerebellar atrophy, ascites or hydrops, pseudo-meconium ileus, renal, pleural or pericardial effusions, hepatosplenomegaly, necrotic or cystic or calcified lesions in the brain, liver or placenta [18]); 8 pregnant subjects presenting with maternal anti-CMV IgM antibodies and 18 healthy control subjects. Samples were obtained after completion of written informed consent. Blood samples were either fetal samples obtained via cordocentesis or neonatal umbilical cord blood. A total of 2–5 ml blood for serology and 2–5 ml blood in EDTA for antigenemia assay was collected from pregnant women and umbilical cord of the newborns or fetuses. All
Table 1. Clinical characteristics of patients and their offsprings. Fetus
Newborn
Total
Maternal Ig M (+) Abnormality & IUGR Control
6 16 0
2 9 18
8 25 18
Total
22
29
51
C Informa Healthcare USA, Inc. Copyright
A. S. Ozgu-Erdinc and C. Sen
Table 2.
Characteristics of patients.(numbers are expressed as mean ± SD (range). Abnormality+ IUGR
Maternal CMV IgM (+)
Control
Total
p value
Fetal Pediatr Pathol Downloaded from informahealthcare.com by University of Newcastle on 09/30/14 For personal use only.
Age 23.5 ± 5.6 (17–36) 27.0 ± 4.2 (21–33) 26.9 ± 4.1 (18–34) 25.2 ± 5.1 (17–36) Gravidity 2 ± 1.7 (1–8) 2 ± 0.8 (1–3) 2 ± 1.1 (1–5) 2 ± 1.4 (1–8) Gestational 30.9 ± 7.63 (18–42) 26.4 ± 9.72 (17–42) 38.6 ± 2.9 (31–42) 32.9 ± 8.1 (17–42) Week
ns ns ns
samples were collected at the same time. CMV serology was performed using a commercial Human ELISA assay. The antigenemia assay (Clonab CMV; Biotest, Dreieich, Germany) has four steps: isolation of blood leukocytes; preparation of microscopic cytospin slides (2.0 × 105 leukocytes per slide); immunoperoxidase staining with the use of monoclonal antibodies (Mabs) to CMV IEA and microscopic evaluation and semiquantitative scoring [19]. The first 2 steps were performed in Istanbul University and the slides were stored at −70◦ C wrapped in foil paper and then the remaining steps were performed in Ankara University as previously described [20, 21]. The presence of dark brown nuclear and cytoplasmic staining was considered as a positive reaction. The results were expressed as the number of positive cells per 200 leukocytes [20, 21]. In the current study presence of a minimum of 10% of positive cells was accepted as positivity. Lack of any reaction was accepted as negativity. Statistics Demographic data and the results were presented as mean with standard deviation (SD) or median with range for continuous variables, and as number with percentage for categorical variables. The Student’s (Independent samples) t-test or the Mann–Whitney U-test was used to compare continuous variables as appropriate, and the chi-square test was used to compare categorical variables. Two-tailed p value of
ORIGINAL ARTICLE
Fetal Pediatr Pathol Downloaded from informahealthcare.com by University of Newcastle on 09/30/14 For personal use only.
Diagnosis of Congenital Cytomegalovirus Antigenemia by Immunohistochemical Detection of Immediate Early Antigen A. Seval Ozgu-Erdinc1 and Cihat Sen2 1 Zekai Tahir Burak Women Health Care, Education and Research Hospital, Perinatology, Ankara, Turkey; 2 Istanbul University Cerrahpasa School of Medicine, Division of Maternal Fetal Medicine, Department of Obstetrics and Gynecology, Istanbul, Turkey
Cytomegalovirus (CMV) can be a cause of fetal morbidity and mortality among approximately 1% of pregnancies. With an aim to detect CMV antigenemia among 51 pregnant women with/without clinically diagnosed abnormalities and intrauterine growth retardation (IUGR) maternal and fetal samples either prenatal (n:22) or postpartum (n:29) were obtained between 17–42 weeks of gestation to analyze anti-CMV IgG, IgM antibodies and cytoplasmic or nuclear CMV antigens. Cytoplasmic and nuclear CMV antigenemia was detected among 19.6% and 11.8% of maternal samples. These values were 29.4% and 17.6% for fetal samples. Among both maternal and fetal samples, there was a 100% correlation when IgG and IgM were negative. The correlation for IgG and IgM positivity was not present among maternal samples since cytoplasmic (37.5%) and nuclear (25%) antigens could not be demonstrated in spite of immunity. Cytoplasmic and nuclear CMV antigens were detected within fetal samples from subjects presenting maternal immunoglobulin positivity, clinical abnormality and clinically normal findings (50, 32, 16.7% and 50, 16, 5.6%) respectively. In conclusion, immunocytochemical detection of CMV antigenemia improves CMV infection diagnosis which may be associated with clinical abnormalities/IUGR. Keywords: CMV, antigenemia, diagnosis, congenital CMV
INTRODUCTION Cytomegalovirus (CMV) is the most common cause of congenital infection affecting 0.2% to 2.3% of births worldwide. Furthermore it is the leading infectious cause of sensorineural hearing loss and mental retardation [1, 2]. High rates of congenital CMV infections have been consistently found in populations with a high seroprevalence [3]. Congenital CMV infection rates increase in parallel with maternal seroprevalence due to transmission from maternal nonprimary infection or reactivation [4]. While CMV IgG seropositivity have been detected among 94–99% of healthy subjects CMV IgM have been found to be positive among 0.2–1.4% of pregnant women in Turkey [5–9]. Although it does not correlate with the risk of antenatal infection, the rates of virus excretion via urine and cervical secretion of pregnant women range from 3 to 12% and
Received 19 July 2013; Revised 11 October 2013; accepted 14 October 2013. Address correspondence to Dr A. Seval Ozgu-Erdinc, Zekai Tahir Burak Women Health Care, Education and Research Hospital, Perinatology, Angora caddesi Ergonul Sitesi No:13 Cayyolu, Ankara, 06810 Turkey. E-mail: [email protected]
Fetal Pediatr Pathol Downloaded from informahealthcare.com by University of Newcastle on 09/30/14 For personal use only.
Diagnosis of Congenital Cytomegalovirus Antigenemia
from 11 to 28%, respectively [10]. A study from Canyılmaz et al. showed that cervical CMV excretion rate during the third trimester of pregnancy was 10.75% [10]. Fetal transmission and teratogenicity at birth or in utero is relatively rare, (10%) of CMV antigens in maternal urine. The risk of intrauterine infection largely depends on the time of maternal infection during pregnancy. Primary CMV infection occurs during pregnancy in 2% of women, with intrauterine transmission in approximately 40% of the cases [11]. Approximately 10% of the infected live born neonates have symptomatic disease at birth. In addition, between 7 and 15% of the asymptomatic newborns develop late squeal, particularly sensorineural hearing loss and neurodevelopmental disorders [12]. Most cases are subclinical in early life and some may present as sensorineural hearing loss when starting at school or during screening tests. The diagnosis of congenital infection can be made by demonstration of CMV; by isolation of virus from urine (Culture techniques); by detection of CMV-IgM in blood (Serological tests); detection of CMV antigen (Antigenemia assay) in blood samples and identification of CMV-DNA by PCR from samples [12–15]. Over the past several years, the direct detection of pp65 antigen in blood leukocytes has been successfully applied to the early diagnosis of CMV infections [16, 17]. As the method permits an early diagnosis before the onset of clinical symptoms, either termination of early gestational week pregnancies or targeted preventive treatment using hyper immunoglobulin is possible. Thus due to its frequent observation and potential complications detection of CMV is needed. With an aim to determine the frequency of CMV antigenemia in our center among pregnant subjects and umbilical cord blood we analyzed the presence of CMV by serological and immunohistochemical methods at a time period when PCR technology for detection of CMV DNA was not available. METHODS This cross-sectional study was conducted in the Istanbul University Cerrahpasa School of Medicine, department of obstetrics and gynecology, division of maternal fetal medicine, Istanbul, Turkey. A total of 51 pregnant women and their offsprings’ (22 fetuses and 29 newborn) were included in the study as shown in Table 1. Blood samples were obtained from 25 pregnant subjects with fetal abnormalities suggesting CMV infection (oligohydramnios, polyhydramnios, intrauterine growth retardation (IUGR), echodensities of bowel, liver or ventricle cerebral ventriculomegaly or atrophy, hydrocephalus, microcephaly, cerebellar atrophy, ascites or hydrops, pseudo-meconium ileus, renal, pleural or pericardial effusions, hepatosplenomegaly, necrotic or cystic or calcified lesions in the brain, liver or placenta [18]); 8 pregnant subjects presenting with maternal anti-CMV IgM antibodies and 18 healthy control subjects. Samples were obtained after completion of written informed consent. Blood samples were either fetal samples obtained via cordocentesis or neonatal umbilical cord blood. A total of 2–5 ml blood for serology and 2–5 ml blood in EDTA for antigenemia assay was collected from pregnant women and umbilical cord of the newborns or fetuses. All
Table 1. Clinical characteristics of patients and their offsprings. Fetus
Newborn
Total
Maternal Ig M (+) Abnormality & IUGR Control
6 16 0
2 9 18
8 25 18
Total
22
29
51
C Informa Healthcare USA, Inc. Copyright
A. S. Ozgu-Erdinc and C. Sen
Table 2.
Characteristics of patients.(numbers are expressed as mean ± SD (range). Abnormality+ IUGR
Maternal CMV IgM (+)
Control
Total
p value
Fetal Pediatr Pathol Downloaded from informahealthcare.com by University of Newcastle on 09/30/14 For personal use only.
Age 23.5 ± 5.6 (17–36) 27.0 ± 4.2 (21–33) 26.9 ± 4.1 (18–34) 25.2 ± 5.1 (17–36) Gravidity 2 ± 1.7 (1–8) 2 ± 0.8 (1–3) 2 ± 1.1 (1–5) 2 ± 1.4 (1–8) Gestational 30.9 ± 7.63 (18–42) 26.4 ± 9.72 (17–42) 38.6 ± 2.9 (31–42) 32.9 ± 8.1 (17–42) Week
ns ns ns
samples were collected at the same time. CMV serology was performed using a commercial Human ELISA assay. The antigenemia assay (Clonab CMV; Biotest, Dreieich, Germany) has four steps: isolation of blood leukocytes; preparation of microscopic cytospin slides (2.0 × 105 leukocytes per slide); immunoperoxidase staining with the use of monoclonal antibodies (Mabs) to CMV IEA and microscopic evaluation and semiquantitative scoring [19]. The first 2 steps were performed in Istanbul University and the slides were stored at −70◦ C wrapped in foil paper and then the remaining steps were performed in Ankara University as previously described [20, 21]. The presence of dark brown nuclear and cytoplasmic staining was considered as a positive reaction. The results were expressed as the number of positive cells per 200 leukocytes [20, 21]. In the current study presence of a minimum of 10% of positive cells was accepted as positivity. Lack of any reaction was accepted as negativity. Statistics Demographic data and the results were presented as mean with standard deviation (SD) or median with range for continuous variables, and as number with percentage for categorical variables. The Student’s (Independent samples) t-test or the Mann–Whitney U-test was used to compare continuous variables as appropriate, and the chi-square test was used to compare categorical variables. Two-tailed p value of
