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The natural history of congenital CMV infection is scarcely known in populations with high maternal CMV seroprevalence. This study evaluated birth prevalence, clinical findings at birth, and hearing outcome in infected children from such a population.
Infants consecutively born were screened for the presence of CMV in urine and/or saliva within the first two weeks. Neonatal clinical findings were recorded and infected children were tested to document hearing function during follow-up. A subset of mothers of infected infants was prenatally tested for the presence of anti-CMV IgG antibodies.
Congenital CMV infection was confirmed in 87/8047 infants (1.08%; 95%CI: 0.86–1.33). Seven (8.1%; 95%CI: 3.3–15.9) infants had at least one clinical finding suggestive of CMV infection and 4 (4.6%; CI95%: 1.3–11.3) had ≥ 3 findings of systemic disease. Sensorineural hearing loss was found in 5/58 (8.6%; CI95%: 2.9–19.0) children tested at a median age of 21 months. Bilateral profound hearing loss was observed in two children and hearing threshold was >60 dBHL in all five children with hearing loss, including two children born to mothers with probable non primary CMV infection.
The results of this first large newborn screening study in a population with high CMV seroimmunity provide additional evidence that congenital CMV disease occurs in populations with high seroprevalence rates with a similar incidence of CMV-related hearing loss to that reported in offspring of women from populations in the developed countries with lower rates of seroimmunity to CMV.
Cytomegalovirus (CMV) is a frequent cause of congenital infection in humans in all regions of the world and an important cause of neurological disease and sensorineural deafness in children in the US [1, 2] and in other developed countries [3, 4].
In contrast to most congenital viral infections, congenital CMV infection and disease can occur in children born to women with pre-conceptional immunity [3, 5–8]. The factors that are associated with intrauterine transmission of CMV and the occurrence of fetal, neonatal, or infant disease have not been well defined . However, the incidence of congenital infection depends on the epidemiological characteristics of the population, in particular, the maternal CMV seroprevalence. High rates of congenital CMV infection have been consistently demonstrated in populations with a high seroprevalence . Therefore, it is not possible to simply extrapolate the knowledge acquired from populations of developed countries with low to intermediate seroprevalence to those of developing countries with high CMV seroprevalence. In addition, it has yet to be established whether congenital CMV infection and disease represents a similar public health problem in developing countries as has been identified in North America and Europe.
Only studies that were done without universal screening, enrolled a small sample of selected infants [11–16] or used non-virologic diagnostic methods  have been reported from Latin America. These studies have reported a wide range (0.10–6.8%) of congenital CMV infection prevalence rates and the frequency of symptomatic infection ranged from none to 66.6% of the infants [11, 14, 16].
We conducted a prospective newborn screening study in a representative sample of low income population with high maternal CMV seroprevalence in the southeast region of Brazil  to determine the prevalence of congenital CMV infection and clinical findings in infected infants. The frequency of CMV-related hearing loss was defined.
Mothers and their newborns attending two public hospitals in Ribeirão Preto city, State of São Paulo, Brazil, were studied. Both hospitals provide care for a low income population with no private health insurance. The first maternity (Mater) provides care for low risk parturients. The second hospital, Clinical Hospital of Faculty of Medicine of Ribeirão Preto, University of São Paulo, Brazil (HCFMRP) serves as a referral center for high risk parturients but also provides care for low risk parturients. A 95.7% seroprevalence of CMV infection has been detected among pregnant women from this population . Approximately 9500 babies are born every year in this city, 4200 (44%) of whom are attended at these two maternity hospitals. The study was approved by the Research Ethics Committee of the University Hospital (Processes 9366/2003, and 9145/2004), and written informed consent was obtained from all participants.
Babies consecutively born and present in one of the two hospitals from 7:00 a.m. on Monday to 6:00 p.m. on Friday were selected for the study based on the following criteria: mother’s consent to participate; any maternal condition or type of delivery, gestational age and clinical characteristics at birth; and the possibility of obtaining a saliva or urine sample during the first week of life. Infants born at HCFMRP were screened between March, 2003 and July, 2007. Screening was performed in infants born at Mater during the year of 2004. A total of 85.3% of the babies born during the study period were enrolled.
Gestational age (in completed weeks) was determined either by obstetrical estimation or by pediatric newborn exam . Neonates were classified as small for gestational age (<5th percentile) or adequate for gestational age (≥5th percentile) according to a standard reference curve . Head circumference was measured by the second day after birth and compared with standard values for term and preterm infants . For the 7 infants with head circumference < 2 standard deviations who were also small for gestational age, microcephaly was defined as the head circumference z score adjusted for weight deficit < −2 .
A saliva and/or urine sample was collected from the newborns within 24 hours of life and processed by polymerase chain reaction (PCR) for the detection of CMV DNA [22, 23]. We have previously demonstrated that urine and saliva samples were comparable for the diagnosis of congenital CMV infection . The detection of CMV DNA in the initial sample was considered to be presumptive evidence of congenital infection. Subsequent urine and saliva samples were collected from these newborns within 3 weeks of life and tested for the presence of viral DNA and inoculated into human fibroblast cells for virus isolation for diagnostic confirmation . In all newborns with a positive screening PCR, CMV DNA was found and virus was isolated in both urine and saliva specimens subsequently obtained. All infants with confirmed infection underwent complete physical examination, ophthalmologic evaluation by fundoscopy (performed by a trained ophthalmologist), and cranial computerized tomography (CT) scan. Hearing evaluation was conducted using auditory brainstem response (ABR) during follow up . Hearing evaluations were not performed at birth. Sensorineural hearing loss was defined as an air conduction threshold of >30 decibels (dBHL) on ABR .
Routine maternal screening at delivery included serological testing for human immunodeficiency type 1 virus, and syphilis. However, when findings suggestive of congenital infection were observed, other congenital infections were excluded, such as those caused by T. gondii, rubella virus, and, in selected cases, Parvovirus B19, enterovirus, and herpes simplex virus. Collection of blood specimens for white blood cell count, platelet count, bilirubin, and liver function tests was performed at the discretion of the child’s physician. For a subset of 44 (50.6%) CMV-infected infants, an available stored prenatal maternal sample was tested for the presence of anti-CMV IgG (VIDAS CMV IgG ELISA, Biomerieux , France) and CMV-IgG avidity index (VIDAS CMV IgG avidity, Biomerieux, France).
Once other congenital infections were excluded, infants were classified as symptomatic based on the presence of at least one of the typical findings suggestive of congenital infection including petechiae, cholestatic jaundice (conjugated bilirubin > 2.0 mg/dL), hepatosplenomegaly, purpura, microcephaly, seizures, chorioretinitis, or CT abnormalities (intracranial calcifications, ventriculomegaly, cerebral atrophy and/or malformations).
To determine the rate of congenital CMV infection among all live born infants, the sample size was estimated based on a previous smaller study that reported approximately 2% prevalence of congenital infection in this population . The sample size necessary for a 99% confidence level, a precision of 0.5%, and assuming α=1% was estimated to be 5176 newborns. To determine the proportion of symptomatic cases among CMV-infected infants, the sample size was increased by 50%. The binomial exact 95% confidence intervals (CI) were calculated for birth prevalence rates and proportions. Categorical variables were compared by univariate analyses using the chi square test with α=0.05. Continuous variables were tested with the unpaired t-test. EPI INFO software, version 6.04 was used in these calculations
A total of 8047 infants (1623 from Mater, and 6424 from HCFMRP) born to 7848 mothers (192 twin pairs, 7 triplets) were enrolled into the study. Most pregnant women (98.2%) received prenatal care. Overall, 87 of the 8047 live born infants had a confirmed diagnosis of congenital CMV infection, an overall prevalence of 1.08% (CI95%: 0.86–1.33) which was similar between infants born at Mater (16/1623:0.98; CI95%:0.56–1.59) and those born at HCFMRP (71/6424:1.10; CI95%:0.86–1.39). In addition to younger maternal age, and lower birth weight, a higher frequency of intrauterine growth restriction (Odds Ratio (OR): 3.00; 95%CI: 1.8–5.0) and twin pregnancies (OR: 2.66; 95%CI: 1.3–5.4) were found among neonates with congenital CMV infection as compared to uninfected infants (Table 1). Although the data on maternal tobacco usage, arterial hypertension and/or chronic diseases were not available for mothers of all uninfected infants, these data were available in a subset of 173 mothers of CMV-uninfected infants who were selected as controls by individual matching according to place and week of birth, and gestational age with mothers of CMV-infected children as part of a separate study. A similar frequency of the presence of at least one of the mentioned maternal risk factors for intrauterine growth restriction between the infected and uninfected groups was found (55.2% vs. 48.0%, respectively),
None of the infants had evidence of other congenital or perinatal infections. As shown in Table 2, at least one clinical finding suggestive of congenital infection was found in 8.1% (95%CI: 3.3–15.9) of CMV-infected infants. The overall prevalence of symptomatic CMV infection was 0.09% (7/8047, 95%CI: 0.03–0.18). Features among three infants with one typical finding were: cholestatic jaundice (1), petechiae (1), and cranial CT abnormalities (1). The remaining 2 infants with CT abnormalities also showed ≥1 additional typical finding. The inclusion of intrauterine growth restriction as defining criteria for symptomatic infection would increase the proportion of symptomatic infants to 26.4% (23/87). Four infants (4.6%; CI95%: 1.3–11.3) had 3 or more typical signs consistent with CMV multisystem disease.
At the time of this analysis, 63/87 (72.4%) infected children underwent at least one ABR testing and the median age hearing testing was 21 months (range: 3–63 months). Among these, 5 (7.9%) children had a conductive loss due to middle ear effusion and it was not possible to determine the cochlear function. Sensorineural loss was found in 5 of the remaining 58 children with adequate evaluation, i.e. 8.6% (CI95% 2.9, 19.0). These 5 children were tested at a median age of 24 months (range: 15–50 months). Bilateral and profound hearing loss (>90 dBHL) was detected in 2 infants and the third child had severe (>60dBHL) bilateral loss. Among 2 children with unilateral loss, one had a threshold of 60dBHL and the second of 90dBHL. The remaining 53 (91.4%) children had normal hearing function. Two of 5 (40.0%) infants with hearing loss had ≥ 3 neonatal clinical signs suggestive of congenital CMV infection, one was small for gestational age, and the other two had no other clinical abnormality.
Among 44 mothers of congenitally infected infants whose prenatal serum specimens were available and tested for anti-CMV IgG antibodies (median gestational age of 13 weeks, range: 2–29), 2 (4.5%) were seronegative and had a primary infection during gestation. The remaining 42 mothers were CMV IgG seropositive (95.5%). Of the seropositive samples, one (2.2%) had a low avidity index (13% at 9 weeks of gestation), and 41 had a high IgG avidity index (> 73%). Among these, 20 samples were collected at ≤ 12 weeks, 19 at 13–25 weeks, and 2 at >25 weeks of gestational age. Considering that full maturation of the IgG antibodies or high avidity index can take approximately 12–25 weeks after primary infection [27, 28], a presumed non-primary infection was responsible for 39/44 (88.6%) congenital infections. It was not possible to determine the type of infection in the mother with low avidity index early on gestation or in two with high avidity at > 25 weeks gestation.
One of the neonates born to mothers with a primary infection had a single finding (petechiae), and the remaining infant was asymptomatic at birth. Hearing evaluation was not performed in one of these children and the other had conductive loss. Among infants born to mothers with presumed non-primary infection, 2/39 (5.1%; CI95% 0.6, 17.3) had one neonatal finding (cholestatic jaundice in one and abnormal head CT scan in the other). The infant with abnormal CT scan showed neurodevelopment delay during follow up. Twenty seven infants (69.2%) born to mothers with probable non-primary infection underwent ABR evaluation. Two of these (8%) were asymptomatic at birth but had hearing loss (one with bilateral and the other child with unilateral loss) detected during follow up (at 15 and 50 months). None of the three neonates born to mothers with undetermined type of CMV infection during gestation were symptomatic. However, bilateral hearing loss was found at 24 months in the infant whose mother had low avidity early during gestation. For the 4 infants with findings of CMV multisystem disease at birth, prenatal samples were not available for testing.
Despite the recognized importance of congenital CMV infection in the world, only limited information is available about the incidence and natural history of this infection in Brazil [11, 12, 14, 15, 17]. In this population based newborn screening study, we found a 1.1% (95%CI: 0.86–1.33) birth prevalence of congenital CMV infection and 8.1% (95%CI: 3.3–15.9) of infected infants exhibited at least one clinical finding suggestive of congenital infection in the newborn period while 4.6% (CI95%:1.3–11.3) had multisystem CMV disease. Our findings are in agreement with previous studies that have demonstrated a significant rate of congenital HCMV infection in infants born to seroimmune women. Although samples were not available from all 87 women who delivered infected infants, analysis of prenatal serum specimens from 44 of these women revealed that 42/44 (95.5%) had IgG antibodies reactive with CMV in a commercial ELISA, a rate of CMV seroprevalence that is similar to our previous study in this population . Furthermore, CMV seroreactivity may underestimate the actual prevalence of infection [29, 30]. Thus, it is conceivable that in this population the rate of CMV infection is >95.5% and that non-primary maternal infection accounts for >95% of infants with congenital CMV infection.
The rate of congenital CMV infection observed by us in infants born to Brazilian women from a predominantly low socioeconomic level is similar to that seen in other highly immune populations in Africa (0.9–1.4%) [31, 32]; Chile (1.8%) ; Korea (1.2%); Mexico (0.9%) ; China (1.8%) ; India (2.1%) , and low income North American women (1.2%) [5, 36]. However, it is higher than that observed in populations with low or intermediate seroprevalence from European [37, 38] or North American [39, 40] populations in which the prevalence of congenital CMV infection ranges from 0.18% to 0.48%. The findings of our study are consistent with previous studies and a recent review of the literature documenting that the incidence of congenital CMV infection increases with increasing maternal CMV seroprevalence .
It is of interest that infected infants were significantly smaller at birth and were almost 3-times more likely to exhibit intrauterine growth restriction (IUGR) than infants without congenital CMV infection. Berge et al. have previously published that congenital CMV infection resulted in significantly lower mean birth weight in low income populations . More recently, it has been suggested that CMV infects the uterine wall and/or the adjacent placenta impairing critical aspects of syncytiotrophoblast and cytotrophoblast differentiation [42, 43], thus altering their capacity to provide oxygen and nutrients to the developing fetus. Even though approximately half (55.2%) of CMV-infected infants of our cohort were born to mothers with other chronic conditions that could result in poor placental function, oxidative stress, and intrauterine growth restriction, the effect of CMV infection on placental function may have also contributed to growth restriction. This study was not designed to test this hypothesis and therefore, complete data on maternal risk factors for IUGR were not obtained for mothers of uninfected infants. However, information about maternal smoking and other chronic diseases was collected in a subset of mothers of uninfected children and compared to the data from mothers of infected infants. This comparison showed that the two groups are similar with respect to the presence of at least one maternal risk factor suggesting that IUGR in infected infants is in part due to placental CMV infection. Although it has been described that in twin pregnancies, infection of only one or both twins can occur  independently of placenta type , ours is the first report suggesting an increased frequency of congenital CMV infection in twin pregnancies.
The rates of symptomatic infection vary considerably among the published reports primarily because of the lack of uniform definition that is consistently applied by different investigators . We opted to classify infants as symptomatic based on the presence of clinical findings that have been consistently described in larger series of more severely affected infants [1, 46, 47]. However, other authors  included the finding of SGA as a typical clinical feature of congenital CMV infection. In the current study, we elected not to classify infants as symptomatic based on the presence of intrauterine growth restriction alone. In addition, by adjusting head circumference for weight deficit, we chose not to classify seven SGA infants as having microcephaly based on standard growth curves. Given that one fourth of the infected infants studied by us were SGA, as many as 26.4% of them would have been categorized as symptomatic if this criteria was included in the definition. Significant findings of our study were the demonstration that, among infants born to mothers with probable non-primary CMV infection, hearing loss greater than moderate severity occurred in 2 (8%) children, and abnormal CT scan with neurodevelopmental delay was found in one child. This is the first study to demonstrate the occurrence of CMV-related hearing loss and neurological impairment in infected children following non-primary CMV infection in a population with very high (95%) seroimmunity to CMV. Our findings clearly document the importance of congenital CMV infection as a cause of morbidity and disability even in populations with high maternal CMV seroprevalence. Since sensorineural hearing function was not assessed in the newborn period and the infected children were not monitored with periodic audiologic evaluations, the frequency of hearing loss at birth and the exact age at which hearing loss occurred could not be determined.
In conclusion, a 1% prevalence of congenital CMV infection and disease was identified in this Brazilian population of predominantly low socioeconomic level. Given the frequency of CMV disease in the newborn period and the CMV-related hearing loss during follow-up, this infection is likely to have a significant impact on the health and quality of life of these children, as demonstrated in other populations from developed countries in North America and Europe. Our results provide additional evidence that congenital CMV disease occurs in populations with high seroprevalence rates, and confirms the findings from previous reports that symptomatic infection and sequelae such as hearing loss also occurs following non-primary maternal infection [3, 5].
We are grateful to Lauro J. Marin and for technical assistance with laboratory assays.
Sources of Financial Support: Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP # 02/04166-6), Brazil and the NIH, NIAID (AI49537) and the Fogarty International Center (R03 TW006480) to WJB.
No authors have any conflict of interest to disclose.