Congenital CMV infection : Prevalence in newborns and the impact on hearing deficit

Congenital cytomegalovirus (CMV) infection is asymptomatic in 90% of infected newborns but approximately 10–20% of these infants are at risk of developing sequelae later, mostly hearing deficit. The aims of the study were to investigate the prevalence of congenital CMV infection in a Swedish population of newborns and investigate the relative risk of hearing deficit in newborns with congenital CMV infection. The dried blood spot (DBS) samples of 6060 newborns in southern Stockholm during 12 months (October 2003–June 2004; August 2004–October 2004) were analysed for CMV DNA by TaqMan based real-time PCR. Hearing deficit was assessed by otoacoustic emission (OAE) within a newborn screening programme. 12 infants out of 6060 or 0.2% (95% CI 0.1–0.3%) had congenital CMV infection. One boy among the 12 infected infants had unilateral hearing loss, indicating that the risk of hearing loss is greatly increased (about 20 times) in CMV infected infants. No child developed ocular complications such as chorioretinopathy during 3 y of follow-up. Congenital CMV has an impact on child health but can easily be overlooked due to lack of signs in the neonatal period. Surveillance for congenital CMV is important in addition to programmes for prevention and treatment.

Congenital cytomegalovirus (CMV) infection is the most common intrauterine infection and 1 of the most important causes of deafness in childhood [1], [2], [3], [4], [5], [6], as well as an important cause of neurological disabilities [1], [5], [7], [8], [9] and visual impairment [8], [10]. Symptomatic infants often present with hepatosplenomegaly, petechial rash, and/or neurological manifestations [5], [9], [11]. However, about 90% of infected newborns have no overt signs of infection [5], [6], [7], [8], [9], [12] and therefore congenital CMV infection is easily overlooked. Even neonatally asymptomatic infants are at risk of developing sequelae later in about 10–20% of cases [1], [5], [6], [7], [9], [11], [13]. The most common sequela is hearing loss which may have late onset [3], [4], [13] and can occur during the first 6 y of life [3]. Moderate or severe impairment has been reported in 22% of the eyes of children with symptomatic congenital CMV infection. The visual impairment was most often caused by an optic atrophy, chorioretinal scars and/or affection of cerebral visual pathways [10]. Visual impairment as well as strabismus occurred significantly more often in symptomatic patients than asymptomatic patients [10].

Epidemiological conditions appear to vary from country to country. The prevalence of congenital CMV has been reported to be between 0.2% and 2.5% [5], [7]. Two recent systematic reviews have reported an estimated average birth prevalence of congenital CMV infection of 0.65–0.7% [7], [8]. Factors that influence the rate of congenital CMV infections are socioeconomic status, breast feeding, stay at day-care centres and seroprevalence of CMV in fertile women [1], [6], [7], [8], [14], [15].

Since the last epidemiological investigation in Sweden in the late 1970s [5], it is reasonable to assume that the incidence of CMV infection might have changed due to increased immigration and changes in lifestyle. Updated epidemiological data are needed in order to be able to provide guidelines regarding prophylaxis and treatment strategies.

Detection of CMV DNA in dried blood spot samples (DBS) is a feasible method for diagnosing CMV and has previously been shown to be a method with high specificity and sensitivity [16], [17], [18]. This method allows screening of neonates in large series using already available samples and is also a tool for retrospective diagnosis in children with hearing deficit, visual impairment or neurological disabilities [9], [17], [18], [19], [20].

The main objectives of the present study were to evaluate the prevalence of congenital CMV in newborns in the Stockholm area and to investigate the relative risk of hearing deficit in newborns with congenital CMV infection.

During the study period (October 2003–June 2004; August 2004–October 2004), 6199 infants were born at 2 different hospitals in the southern part of Stockholm (Karolinska University Hospital, Huddinge and Södertälje Hospital). The parents of 139 (2%) infants declined to participate. We used the DBS sample, which is routinely collected at the age of 3–5 d for screening of inherited metabolic diseases, to detect CMV DNA after informed consent from the parent(s). All cards were analysed with quantitative CMV DNA PCR (TaqMan). Positive cases were analysed in triplicate.

The study was performed with the consent of the ethics committee at Huddinge University Hospital.

All of the children with CMV DNA identified in the DBS were contacted by the same paediatrician (M-LE) and the diagnosis was confirmed by viral culture from urine, serology and PCR analysis in blood. In order to evaluate the extent of the disease, blood cell count and liver enzymes were analysed in blood samples and head ultrasound was carried out with the consent of parents.

Two discs from each card were used for the extraction of DNA. The discs were placed in a tube with 50 microlitres of MEM (GibcoBRL/Life Technologies, UK). The samples were then incubated at 56°C for 1 h and at 95°C for 10 min. After a rapid cooling, the samples were centrifuged for 5 min at 14,000 r.p.m. and the supernatant from each sample was moved to new tubes and placed at −70°C [17].

The method has been described earlier [21]. In brief, the PCR reaction is based on primers and probe from the pol region and was performed in a 96-well optical reaction plate under the following conditions: 50°C for 2 min; 95°C for 10 min; 50 cycles of 95°C for 10 s and 58°C for 15 s. The number of threshold copies/volume was set to be at least 1 after at most 50 cycles. The final amplified product was a 66 base pair fragment. The human albumin gene was used as a control of DNA extraction to detect inhibition causing false negative results.

All cases of stillbirth (from 22 gestational weeks) are routinely investigated for congenital CMV infection at the hospitals included in the study. In these cases, CMV serology is performed in the mother and the placenta is examined for CMV by the PCR technique.

The children were assessed with regard to growth, hepatosplenomegaly, petechial rash and jaundice in the newborn period and monitored subsequently with respect to developmental milestones up to 3 y of age.

Of 12 infants identified with congenital CMV infection, 11 underwent repeated ocular assessments. The assessment was adapted to the age and cooperativeness of each child. One child declined ocular examinations. Visual acuity was assessed with Teller acuity cards (Vistech Consultants, Inc.) or by testing the child's ability to fixate and follow small objects. In older children, best corrected decimal visual acuity with optotypes was assessed. Other evaluations included tests of ocular motility, binocularity (Lang, Forch, Switzerland) and cover test at near to detect strabismus. Retinoscopy and indirect ophthalmoscopy were performed after administration of cycloplegic eyedrops (0.5% cyclopentolate and 0.5% phenylephrine instilled twice in infants with a body weight < 10 kg, and 0.85% cyclopentolate and 1.5% phenylephrine instilled once in children with a body weight > 10 kg). All children were examined by the same paediatric ophthalmologist (KTF).

In a bedside universal hearing screening, newborns are investigated by transient-evoked otoacustic emissions (TEOAE) at the hospitals included in the study. TEOAE were recorded in the non-linear quickscreen mode with ILO 288 (Otodynamics Ltd.) according to the following pass criteria: whole wave reproducibility ≥70%, signal-to-noise ratio (S/N) ≥ 3 dB in at least 3 of the upper 4 wide frequency bands provided by ILO instrument (centre frequencies from about 1 500 to 4000 Hz + / − 400 Hz b.w.) and at least 50 sweeps.

Hearing in CMV infected children was further assessed with behavioural audiometry at 1 y of age (±3 months) and play audiometry at 4 y of age (±6 months). The majority of assessments were performed at the audiological clinic by a qualified audiologist, but 2 of the children were only assessed by a trained nurse at the well-baby clinic at 4 y of age.

Maternal infections were categorized by comparing stored pre partum samples obtained in week 12 with post-delivery sera obtained at inclusion. Primary maternal infection was defined by evidence of de novo seroconversion between the samples or the presence of CMV-specific immunoglobulin M (IgM) antibodies during pregnancy. Maternal infection was presumed to be recurrent when CMV-IgG specific antibodies without IgM were present in the serum from the first trimester and an elevation of IgG antibodies could be identified when comparison was made with the sera from the first trimester.

1 000 serum samples (every 100th) from pregnant women in the population were determined for IgG antibodies against CMV by an earlier described Elisa assay [22]. Purified nuclear CMV antigen (AD 169) was used and the cut-off level for seropositivity was an absorbance of ≥0.2 at a dilution of 1/100.

The odds ratio between CMV and non-CMV subjects for hearing deficit was estimated using logistic regression and its corresponding 95% confidence interval. Odds ratio equal to 1.0 was tested using the Wald statistic. An odds ratio greater than 1 is interpreted as increased risk for hearing deficit in the CMV group. Odds ratio equal to 1.0 indicates no difference between CMV and non-CMV group. p-values < 0.05 were considered as statistically significant.

12 infants were identified with CMV DNA-positive DBS and CMV-positive viral culture from the urine (Figure 1). 10 of these infants had a verified congenital CMV infection. The infections in the remaining 2 infants were classified as possible congenital infection due to CMV DNA only in 1 out of 3 PCR runs in the analysis of the DBS sample in combination with confirmatory urine sample beyond the time frame of 3 weeks (Table I). In 1 of the infants (C) the mother had seroconverted between the first trimester and the post-delivery sera taken when the child was 2 y of age. The mother of the second infant (M) had a rise in IgG antibody titre between pre- and post-delivery sera.

Graph: Figure 1. Results of the CMV screening.

Table I.  Data of virological assessment and diagnosis of infection in mother and child with confirmed congenital infection.

1112 M: mean value of DNA copies in the different PCR runs.

In 9 additional cases we found copies of CMV DNA on the DBS cards but the viral culture and PCR from the urine was negative. None of the children in this group had any clinical symptoms. The mother of 1 infant was seronegative post partum, which excludes the possibility of congenital infection in that infant. In order to reduce the risk of false negative results, every hundredth negative sample (n = 62) was reanalysed and they were all found to be negative.

In 6039 DBS samples CMV DNA was not detected. However, in 27 of these the albumin gene was negative indicating that there was too little material to amplify or there was a possible inhibition.

No case of stillbirth was positive in the examination for CMV during the study period.Requiring both criteria of congenital CMV infection to be fulfilled (positive PCR and confirming positive urine culture) we estimated the CMV prevalence to be 0.2% (95% CI 0.1%–0.3%).

Clinical data of the congenitally infected children and demographic data of the mothers are presented in Table II. No infant had overt signs of CMV disease at birth. One boy (F) had unilateral hearing deficit diagnosed by OAE within the universal hearing screening programme in the neonatal period and confirmed by auditory brainstem response at 1 month of age. This child had normal neuroradiographic imaging (computed tomography). CMV DNA in the cerebrospinal fluid was negative but abnormal cerebrospinal fluid indices with monocytosis were noted. His mother had a recurrent infection during pregnancy. Six other children (D,E,H,I,J,K) were assessed with ultrasound and no abnormalities were found.

Table II.  Demographic data of the mothers and clinical data of congenitally infected infants.

1113 Nationality: S: Swedish; NS: non-Swedish. Profession: D: day-care; H: hospital; A: administrative work. OAE (otoacoustic emission): N: normal; P: pathological. Hearing assessment: N: normal; P: pathological; –: not assessed. Ophtalmological assessment: N: normal; –: not assessed.

The birth weights of the infants were appropriate for gestational age and all children had a normal head circumference. Three of the infants were premature, born ≤37 weeks gestation (Table II). In 1 of the cases born at gestational age 34 weeks (child D) the prematurity can be explained by twin gestation. Only 1 of the twins was congenitally infected. In the 2 other cases, born at gestational week 36 (child G) and 35 (child H), respectively, the congenital CMV infection might have been involved in their preterm birth. Child H had transient elevated gamma glutamyl transferase.

Blood samples were collected in 10 infants at enrolment. Whole blood for the analysis of PCR was available for 7 infants. Of these, 5 were viraemic as determined by CMV DNA in the blood. Serum for analysis of antibodies was available in 9 cases. In 7 (A,B,E,F,G,H,J) of these, IgM antibodies were detected. Five of the infants had intermittent slight laboratory abnormalities with regard to liver function. Transient elevated levels of alanine aminotransferase were found in 3 infants (E, K, L) and of gamma GT in 3 infants (E,H,L).

In 1 case (A), the parents refused further follow-up but they reported that the child was doing well at 1 y. At the 3-y follow-up all children had developed normally with regard to developmental milestones.

The infected children underwent ocular assessments 2–8 times (median 3) during a mean follow-up period of 2–3 y (range 0.7–3.6 y). Median age at latest assessment was 3.1 y (range 0.9–4.0 y). All children presented a visual acuity above 0.63 binocularly (or 0.5 monocularly) or a Teller acuity cards result that was normal for age at latest follow-up. A significant refraction error (hyperopia-astigmatism) was present in 1 child. This child also had a strabismus (exotropia) and was prescribed eyeglasses and patching due to anisometropia. Five children had slight hyperopia (in 1 child associated with astigmatism) but were not prescribed glasses. None of the 11 children assessed with ophthalmoscopy had any signs of active chorioretinitis, inactive chorioretinal scars or optic atrophy.

During the study period 6119 newborn infants were screened by OAE. Of them 27 (0.4%) had either unilateral or bilateral hearing deficit. One of the infants with unilateral hearing deficit had congenital CMV infection. This boy had no family history of hearing deficit and there was no evidence for any other causative agent of the hearing deficit. Odds ratio for hearing deficit in CMV infected infants compared with uninfected was 21.3 (95% CI 2.6–170.8; p = 0.04).

Hearing was assessed in 10 of the infected children at 4 y of age. One child was assessed at 2 y of age but did not participate in the follow-up at age 4 y. The parents of the remaining child had declined to participate in the study follow-up (Table II). Hearing was normal at the 20dB level in 9 children at follow-up. The boy with unilateral sensorineural hearing deficit in the newborn screening had unilateral hearing deficit at 70–85dB at 4 y of age. Another child with normal hearing in the newborn screening had unilateral hearing deficit at 20–35dB but also signs of otosalpingitis at assessment.

Seven out of the 12 mothers whose infants had a confirmed CMV infection had a primary infection during gestation. Four mothers had signs of recurrent infection with increased IgG titre in the sample collected after delivery compared with the sample from the first trimester. One mother had a stable IgG titre between the 2 collected samples.

Pre partum and post partum sera were available from 3 mothers whose infants had copies of CMV DNA on the DBS but negative viral culture from the urine. One of the mothers was seronegative for CMV and 2 had a stable IgG titre between the samples.

Demographic data of the mothers revealed that 8/12 were born in Sweden. The mean maternal age was 32 y. Nine of 12 mothers had documented contact with toddlers during pregnancy, which is a documented risk factor for CMV infection [6]. In 7 cases the infected infant had an older sibling. In addition, 2 of the primiparous mothers worked at day-care centres.

72% of the mothers of the 6060 infants in the cohort had CMV IgG in the sample collected from the first trimester as a sign of past infection.

Congenital CMV infection constitutes a major public health burden [1], [5], [7], [9], [10], [13], [20]. The clinical impact on hearing loss, visual impairment and neurological damage underscores the need for epidemiological surveillance and diagnostic methods with high diagnostic power. The latest Swedish epidemiological survey was performed in late 1970s and showed that 0.5% (95% CI 0.4%–0.6%) of all newborn infants shed CMV virus in urine [5]. Approximately 10% of these infants developed hearing deficits or neurological impairments even if they had no signs of congenital infection in the neonatal period [5].

In order to provide an update of the Swedish epidemiological baseline data, we performed a survey of 6060 DBS cards from newborn infants in 2 different hospitals in the Stockholm area. We identified 12 infants with positive DBS and positive CMV viral culture from the urine. This indicates a prevalence of 0.2% (95% CI 0.1%–0.3%).

In addition to these 12, we also found 9 cases with copies of CMV DNA on the DBS card but without CMV in the urine. None of the infants in the last group had any clinical symptoms of CMV infection, so what can be a possible explanation for these cases where copies of CMV DNA were found but the viral culture was negative? The viraemic phase of congenital CMV is not well studied. Demonstrable viraemia at the DBS sampling is supposed to be related to congenital infection but might also represent infection later. Another explanation could be that the specificity of the test is somewhat low. In fact, recently the need to improve its specificity and sensitivity has been discussed [18]. Barbi et al. suggest an algorithm, meaning that the first test has to be performed on 3 series of punches, 2 of which have to be positive for a congenital infection to be considered. Otherwise, 1 amplification in a first series of 3 has to be positive, and a second in a further series of 3 has to be positive [18]. It was not possible to test each DBS sample 6 different times in 6 different (CMV DNA) runs in our study since we only had access to a very limited amount of material (DBS). In order to reduce uncertainties we reanalysed each 100th negative sample and we could confirm that they were all negative. It remains to be further evaluated whether newborns with CMV DNA identified in the DBS and negative CMV viral culture from the urine are false positives or have any clinical significance. Until this has been clarified the combination of different diagnostic tools is valuable and should be used whenever possible. The prevalence of congenital CMV infection identified in the present study is lower than the prevalence reported in the 1970s. However, a limitation of the comparison is that different diagnostic tools have been used in the 2 epidemiological studies. In the former study the diagnostic tools were viral culture of the urine and analysis of CMV IgM in cord blood. We used CMV DNA PCR analysis of DBS samples for diagnosing congenital infection due to the feasibility and earlier proven good sensitivity and specificity of the test [17]. Studies using PCR instead of viral culture have been shown to report lower birth prevalence [8], however, and it cannot be ruled out that the difference between the 2 studies is due to an underestimation of congenital CMV infection in the present study. For instance, it is possible that infections early in gestation no longer exhibit viraemia when the infant is born.

The use of DBS screening offers possibilities for large-scale epidemiological research, however, and it would not have been possible to screen a population of 6060 infants with virus isolation of the urine due to logistic concerns and ethical principles. The strength of this study is that the vast majority (98%) of infants in the studied population were included in the screening. A shortcoming of the study design was that we did not manage to confirm the diagnosis with urine sample within 3 weeks in more than 4 infants.

The seroprevalence among fertile women in Sweden has not changed since the 1960s. It is rather high (72%) compared to other high-resource countries [6]. This means that secondary infections play an important role in congenital infection and morbidity among children in Sweden. The morbidity is supposed to be more severe after primary infection and there is evidence that the risk of severe brain abnormalities like migration disturbances, ending up in severe disabilities, is higher during the first and second trimester, probably most often after a primary infection [9], [19], [20]. On the other hand, secondary infections are considered to result more often in audiological or milder neurological sequelae [12]. However, there are conflicting findings on this subject [5]. In our study we found that 5 out of 12 infants were infected due to secondary infections. In this category we found the only symptomatic infant at birth. This infant had unilateral sensorineural hearing loss. The finding of 1 infant with hearing deficit out of 12 infected infants is in accordance with other studies [4], [13]. The results also show that 4% (1/27; 95% CI 0.1%–19.0%) of the hearing deficits in this cohort were caused by congenital CMV infection. Furthermore, results indicate that the risk of hearing deficit is 21 times higher in infants with CMV infection compared with uninfected infants. One child with normal hearing in the newborn screening had mild hearing deficit at 4 y follow- up, most likely due to otosalpingitis, but late-onset CMV related hearing deficit cannot be ruled out.

Ocular complications related to congenital CMV infection were not apparent among the 11 infected infants who were followed up. This could be due to the fact that the infants were asymptomatic at birth [10]. Nevertheless, we have observed several other children with congenital CMV and chorioretinopathy and/or cerebral visual dysfunction at our clinic and therefore we strongly recommend regular visual and ocular assessment of all children with congenital CMV infection.

Congenital CMV infection has a great influence on child health. Development of a successful CMV vaccine is considered to be of high priority on the basis of child disability and cost that would be alleviated. Experimental vaccines include a live attenuated vaccine and a subunit vaccine [23]. Until a safe vaccine is available, efforts for the development of prevention strategies such as hygiene measures [4], [15] and successful treatment programmes to limit the risks for long-term sequelae due to congenital CMV, are important issues [8]. Frequent contact with asymptomatic toddlers who secrete virus in the urine is 1 of the most important sources of maternal infection during gestation [1], [6], [14], [15]. In this cohort 7 out of the 12 infants had an older sibling. Furthermore, among mothers who were primiparous, 1 mother with primary infection (A) worked at a day-care centre during pregnancy. These findings could have implications for preventive measures such as frequent hand washing during pregnancy.

Currently, no standard antiviral treatment is available in clinical practice to treat congenital CMV but research is underway to evaluate the risk/benefit ratio of different treatment strategies in infants younger than 3 months with symptomatic congenital CMV infection [6]. Preliminary results indicate that infants with hearing loss and CNS abnormalities may benefit from treatment with gancyclovir [2]. However, the use of gancyclovir for treatment is, at the moment, limited to seriously ill infants due to the toxicity of the drug [6], [15]. Sequelae after a teratogenous influence such as migration disturbance during early brain development are unlikely to be influenced by antiviral therapy. Recently, hyperimmune globulin has been used in an uncontrolled trial for prevention and treatment of foetal infection in pregnant women with primary CMV infection during gestation, with seemingly favourable effect [12], [24]. However, the findings have to be repeated and further evaluated in randomized studies.

Early detection of hearing deficit is important for language acquisition and also for social development in the child [25]. Congenital CMV adds considerably to the morbidity of hearing deficit in infants. Our results indicate that the risk for hearing deficit is greatly increased in CMV infected infants. To date, no child in our study had confirmed late-onset hearing loss, but late onset has been reported in several other studies [3], [4], [13]. In this regard CMV screening combined with OAE screening would make early detection of hearing deficits possible, as well as appropriate interventions, such as early linguistic stimulation, sign language and cochlea implantation. CMV DNA detection on DBS sample is well suited for screening purposes due to the feasibility, but as indicated by the present study specificity and sensitivity have to be further evaluated before screening can be implemented. Another important issue that has to be considered is the risk of unnessessary parental anxiety caused by positive screening results in infants who will never have any manifestations of disease or sequelae.

The study was supported by research grant funds from Samariten Foundation, Sunnerdal foundation, Johan and Linnea Karlsson foundation, Margit Thyselius Foundation for Blind Youth, Svenska Läkaresällskapet and Karolinska Institute, Stockholm, Sweden.

Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.