Fetal brain imaging provides valuable information in cCMV infected infants

Abstract Background Congenital CMV infection (cCMV) is the most common congenital infection with 10–15% of cases developing symptomatic disease. Early antiviral treatment is of essence when symptomatic disease is suspected. Recently, the use of neonatal imaging has been implicated as a prognostic tool for long term sequalae among asymptomatic newborns at high risk. Even though neonatal MRI is commonly used in neonatal symptomatic cCMV disease, it is less often used in asymptomatic newborns, mainly due to cost, access and difficulty to perform. We have therefore developed an interest in assessing the use of fetal imaging as an alternative. Our primary aim was to compare the fetal and neonatal MRIs in a small cohort 10 asymptomatic neonates with congenital CMV infection. Methods We performed a single-center retrospective cohort study (case-series) on a convenience sample of children born from January 2014 to March 2021 with confirmed congenital CMV infection who had undergone both fetal and neonatal MRIs. We created a checklist of relevant cerebral abnormalities and asked 4 blinded radiologists to assess the MRIs (2 for each, namely fetal and neonatal) and then compared the findings between the fetal and neonatal imaging as well as the concordance in reporting of abnormalities within each category. Findings Overall concordance between prenatal and postnatal scans was high (70%). When comparing the two blinded reports for each MRI, we found high levels of concordance: 90% concordance for fetal MRIs and 100% for neonatal MRIs. The most common abnormalities identified in both fetal and neonatal scans were “abnormal white matter hyperintensity” and “subependymal cysts.” Interpretation Even though this is a small descriptive study, it indicates that fetal MRI could potentially provide us with similar information as neonatal imaging. This study could form the basis for subsequent larger future studies.


Introduction
Congenital CMV infection (cCMV) occurs with a worldwide prevalence of 0.2-2.5% and is therefore considered the most common congenital infection [1]. Even though most children with cCMV are asymptomatic, 10-15% of cases develop symptomatic disease leading to thousands of children born every year with permanent neurological disabilities such as sensorineural hearing loss (SNHL) [2], visual impairment and cognitive deficits. Despite its clinical significance and the substantial burden of disease related to cCMV, it still goes undetected due to absence of screening programs at birth and low awareness among pregnant women [3]. Additionally, most of infected newborns are asymptomatic at birth while prognostic biomarkers for disease severity are lacking [4].
Over the past decade, advances have been made with regards to therapeutic strategies. Specifically, beneficial outcomes have been shown on preventing hearing deterioration with the early use of valganciclovir in children with cCMV [5]. With the prospect of new viable therapies, accurate and timely diagnosis has become paramount [6]. Importantly, the dichotomous definition of symptomatic versus asymptomatic newborn may need to be revised since it is apparent and remains challenging how to identify asymptomatic newborns that are at risk of developing SNHL and/or neurological sequalae and therefore might benefit from early antiviral treatment [7]. To date, there are no reliable perinatal biomarkers of disease severity [8].
Recently, the use of neonatal imaging (MRI and US) has been implicated as a potential prognostic tool of symptomatic disease [9]. It has become clear, that despite the current difficulties in assessing the clinical relevance of each imaging abnormality, normal neonatal imaging has a high negative predictive value for poor postnatal clinical outcome [10]. Recent evidence suggests that combining MRI and cranial ultrasound is important in order to obtain a more accurate evaluation of CNS involvement in children with cCMV [11]. Furthermore MRI abnormalities at birth have been proven to be an independent risk factor for long term sequalae in children with cCMV [12].
MRI is widely used in infants. However, limitations such as getting access to the MRI and the anesthesiology team that will take part in performing an MRI scan in neonates exists. Moreover, in many areas of the world, sedation is still required, introducing more ethical dilemmas for both physicians and parents, especially when discussing imaging of asymptomatic newborns [13]. On the other hand, fetal MRI is less invasive and occurs at a prenatal stage providing more time for decision making. In this retrospective case series, we included cases of children with confirmed cCMV who had undergone both fetal and neonatal MRIs. Our aim was to (1) compare the ability of fetal versus neonatal MRIs to recognize cerebral abnormalities related to cCMV infection; (2) record the cerebral abnormalities in the fetal and neonatal MRI of each cCMV case; (3) compare the concordance in reporting of abnormalities by two blinded esteemed radiologists within each category.

Study population and setting
We performed a single-center retrospective cohort study on a convenience sample of newborns born from January 2014 to March 2021 with confirmed congenital CMV infection post maternal primary CMV infection during early pregnancy (<20 weeks) who had undergone both fetal and neonatal MRIs. Although there are no official guidelines, most obstetricians in Greece test for CMV during the first prenatal visit.
Maternal primary CMV infection diagnosis was made post either seroconversion or detection at the first prenatal of CMV-IgM with a low IgG avidity and/or CMV viremia. All mothers underwent amniocentesis that confirmed fetal infection. Fetal MRI was performed due to high risk of developing symptomatic cCMV disease with seroconversion occurring prior to the 20th week of pregnancy, although no CNS findings were noted in fetal ultrasound. A confirmed case of congenital CMV was defined in newborns with a positive urinary CMV PCR (polymerase-chain-reaction) in the first 2 weeks of life. cCMV infected infants who had undergone an MRI after the age of 3 months were excluded. Parents of children followed in our center have provided informed consent for the anonymous use of the data, since all demographic, clinical and therapeutic data are introduced in the European collaborative registry (cCMVnet). Since this is a retrospective cohort study using anonymous data, new informed consent was waived.
Maternal age and timing of maternal CMV infection were recorded, as well as administration of CMV-HIG during pregnancy. Moreover, neonatal clinical data (GA at birth, somatometric measurements, symptoms), laboratory and imaging abnormalities and receipt of antiviral treatment were noted. cCMV infected newborns were considered symptomatic if they presented with clinical findings suggestive of cCMV such as microcephaly, hepatosplenomegaly, petechiae and/or if they had SNHL or neuroimaging findings suggestive of CNS involvement [14] Data collection and procedures MRI images from the 10 cases were sent to the participating radiologists for interpretation. Specifically, four esteemed consultant radiologists with expertise in fetal and neonatal imaging took part in the study in order to assess MRI scans. Two pediatric radiologists assessed fetal MRIs and two radiologists (a pediatric radiologist and a neuroradiologist) assessed the neonatal scans. The radiologists were blinded to the clinical outcome of the child, as well as to the other radiologist's interpretation of the scan. In order to facilitate the comparison between the reports, the authors and radiologists agreed on a list of relevant cerebral abnormalities that were going to be assessed prior to looking at the images. Therefore, an MRI was classified as "abnormal" in the presence of any of the following abnormalities: Gyral abnormalities, ventricular dilatation, ventricular adhesions, subependymal cysts, calcifications, "abnormal white matter T2-weighted signal hyperintensity" (parieto-occipital), "abnormal white matter T2 hyperintensity" (temporal), temporal horn dilatation, cerebellar abnormalities, ventricular adhesions-temporal/occipital lobe, temporal/occipital lobe cysts, microcephaly, microencephaly, cerebellar hypoplasia [15]. "Abnormal white matter T2 hyperintensity" refers to areas with a signal intensity higher than that of the adjacent unmyelinated white matter, which is normal for the individual's age [16].

Statistical analysis
Descriptive statistics were used to summarize the data; concordance rates between MRI reports were described as proportions within and between fetal and neonatal MRI scans. We assessed the ability of fetal MRI to predict abnormalities in the neonatal MRI and clinical outcome by calculating the P value through using the McNemars exact test. All analyses were performed using the programming language R and with Microsoft Excel.

Results
We included all children (n ¼ 10, 4 boys) with confirmed congenital CMV infection, monitored in our outpatient infectious diseases clinic with a mean follow up of 45.4 months (median 34 months, range 14-96 months) ( Table 1). At birth, eight newborns had no clinical findings while one baby had SNHL and evidence of chorioretinitis and another presented with mild hepatitis (AST 82 mg/dL). All babies tested had detectable CMV viremia (8/8, mean 35.745 vc/mL) while CMV-DNA was detected in 2/5 CSF examinations. Neonatal blood viral load did not correlate with fetal or neonatal MRI findings. Head ultrasound had findings suggestive of cCMV CNS involvement in 4/10 newborns. Four children received antiviral treatment for 6 months with oral valganciclovir, while one started while in NICU with IV ganciclovir and continued with PO treatment post discharge ( Table 2). In the three asymptomatic newborns antiviral treatment was decided because of abnormal neuroimaging findings after discussion with parents. At present the child with SNHL since birth is 8 years old (patient 1 in Table 2), has evidence of neurodevelopmental impairment requiring therapy and is wearing hearing aid. All other children are well, none has developed SNHL or evidence of neurodevelopmental disability."

Radiological findings
Fetal MRI: concordance between two radiologists for each abnormality In order to evaluate the data, we calculated the concordance for reporting each individual abnormality within the fetal MRIs category (Table 2). Overall, the concordance between the 2 radiologists was high. Specifically, for 9 out of 14 categories of abnormalities the concordance was 100%. Regarding the remaining 5 categories there was a degree of disagreement between the radiologists. For the presence of "gyral abnormalities" the concordance was 50%, meaning that in 5 out of the 10 cases both radiologists agreed on the presence of gyral abnormalities; in the remaining 5 cases, the radiologists disagreed on the presence of this finding. The findings with the least concordance were "gyral abnormalities" (50%), and "abnormal white matter T2 hypersensitivity (temporal)" (60%). However, we calculated a p value¼ .3 which indicates no statistically significant difference between the two radiological reports. An image representing the concordance and discordance between radiologists regarding fetal images is available (supplemental Appendix 1) Neonatal MRI: concordance between two radiologists for each abnormality Similarly, we calculated the concordance in reporting each abnormality within the neonatal MRI category. In 10 out of the 14 categories of abnormalities the concordance rate was 100%, meaning that the radiologists agreed on the presence of those abnormalities throughout the 10 cCMV cases. For the remaining four categories of abnormalities (abnormal white matter T2 hyperintensity-temporal, temporal horn dilatation, cerebellar abnormalities, cerebellar hypoplasia) the concordance rate between the two radiologists interpreting the scans was still high at 90% (Table 3), indicating no difference in reporting between the two radiologists (p ¼ .1). An image representing the concordance and discordance between radiologists regarding neonatal images is available (supplemental Appendix 2) Fetal vs neonatal MRI Overall, the concordance between the two fetal MRI reports for each case was high (90%) with 9 out of 10 cases being classified as "abnormal" by both radiologists (Table 2). For neonatal MRIs, the concordance rate was overall higher compared to the fetal MRI reports. MRI reports were concordant for each one of the cases (100%).
We then compared the two categories (fetal vs neonatal MRI) with regards to the reported abnormalities and whether the radiologists reported the scans to be normal or abnormal (scan outcome) ( Table 2). In 7 out of 10 cases, both were reported abnormal by all 4 radiologists, with a positive predictive value of fetal vs neonatal MRI calculated at 70% (7/10, 95% CI, 35-92). In 2 cases (20%) (case 2 and 5), the fetal MRI was deemed abnormal by both radiologists, but the neonatal were considered normal. In 1 case (case 7), the radiologists evaluating the fetal MRI disagreed on whether the scan was overall "normal" or "abnormal," but both radiologists examining the neonatal MRI agreed that the neonatal scan was normal. An image representing the concordance and discordance between radiologists regarding fetal vs neonatal images is available (supplemental Appendix 3)

Discussion
Despite the worldwide high burden of disease related to cCMV, long-term prognosis is often difficult to predict during the perinatal period since most children are asymptomatic at birth [17]. Although most asymptomatic cCMV infected newborns will remain healthy, an important percentage ranging 10-15% may present sensorineural hearing loss (SNHL) while few children will later develop neurodevelopmental sequalae [18]. Considering the encouraging clinical outcomes when providing rigorous follow up and antiviral therapy to cCMV symptomatic neonates, it has now become imperative to discover prompt and reliable noninvasive biomarkers of disease severity for asymptomatic  Gyral Abnormalities 5 (50) 0 (0) 5 (50) 5 (50) 9 (90) 0 (0) 9 (100) 1 (10) newborns and to tackle the issues related to delayed diagnosis [19]. To date small studies have proposed that high CMV DNAemia [20,21] at birth as well as low birth weight and head circumference may serve as such. Identification of predictive biomarkers of longterm neurodevelopmental sequelae and SNHL among asymptomatic cCMV infected newborns will enable development of clinical trials to examine the impact of antiviral treatment on those at high risk. Until recent years, an MRI scan was usually indicated only in cases with symptomatic cCMV infection or those with abnormal brain ultrasound. However, recent studies highlight the complementary role of the two imaging techniques. Keymeulen et al. published a study showing that 20% of cCMV children with a normal cranial US, had abnormal MRI findings. Of those cases, 91% were classified as symptomatic, 40% of which were graded as severely symptomatic based on MRI lesions alone [11]. Additionally, Lucignani et al. constructed a brain MRI score and suggested that although symptomatic newborns are more likely to present with abnormal MRI, a high score correlates with long-term adverse neurologic outcome in both symptomatic and asymptomatic newborns [22]. Furthermore, a recent meta-analysis indicated that in 6% of fetuses with cCMV and normal fetal neurosonography, cerebral abnormalities were identified exclusively on fetal MRI. These study outcomes support the importance of longitudinal evaluation using fetal MRI in cCMV infection, even when an ultrasound appears to be normal at diagnosis [23,24]. It thus appears that fetal and neonatal brain MRI has utility for treatment decision making on behalf of asymptomatic cCMV newborns [25]. We were particularly interested in evaluating the role of fetal MRI as a predictor of sequelae in cCMV infection. Postnatal imaging, although more commonly used, may often require patient's sedation in order to achieve diagnostic images, raising ethical questions regarding the potential neurotoxic effects of anesthesia on the developing brain [26]. Furthermore, delays in performing the MRI scan due to resources accessibility issues (i.e. MRI scan availability, anesthesiologist) subsequently may result in delay in treatment initiation. On the other hand, fetal MRI occurs at an earlier stage and is noninvasive for both the mother and the fetus. If fetal imaging could provide comparable information to neonatal, the difficulties related to the later could then be avoided. Moreover, fetal MRI could function as a screening tool to minimize the need for neonatal MRI. In this case neonatal imaging could be performed in a subset of children at increased risk.
In this study we present 10 cases of children with cCMV who had undergone both fetal and neonatal MRI. Our primary aim was to compare the fetal and neonatal images with regards to cerebral abnormalities within each case. We created a checklist of relevant cerebral abnormalities and asked two blinded radiologists to assess each MRI. Prenatal and postnatal scans were found concordant in 70% of cases. One of the cases (10%) had partially concordant scans, since the two radiologists reporting on the fetal images disagreed regarding the severity of abnormalities. Two cases (20%) had abnormal fetal MRIs but normal neonatal MRIs (Table 2). Thus, fetal MRI monitoring appears to have sufficient sensitivity for use as a "screening" tool for diagnosing CNS abnormalities. The most common abnormalities identified in both fetal and neonatal scans were "abnormal white matter hyperintensity" both in the parieto-occipital and temporal areas. This finding agrees with previous studies [27]. White matter abnormalities (WMA) are common and potentially important to identify. Abnormal T2 hyperintensities are a characteristic finding in encephalopathy resulting from CMV infection and typically involve deep white matter while sparing subcortical white matter. They are usually multifocal and affect the frontal, parietal, and temporal lobes, with larger lesions often located in the parietal lobe. Congenital muscular dystrophies can present with a similar spectrum of MRI findings in the brain as CMV infection. However, in addition to muscle weakness, these cases are typically associated with pontine hypoplasia and subcortical cerebellar cysts, which were not observed in our patients. Clinically, classic leukodystrophies usually have a progressive course, while encephalopathy resulting from CMV infection is typically clinically static. Furthermore, our population had a positive urinary CMV PCR (polymerase chain reaction) result detected in the first two weeks of life [28].
Recent data suggest that when WMA are not associated with structural lesions such as ventriculomegaly and may represent inflammation subject to antiviral treatment antiviral that targets CMV. This is in contrast to rather static irreversible lesions, such as cysts or gyral abnormalities [29] Indeed, the least concordance (50%) on prenatal MRI interpretation was observed at the evaluation of the presence of gyral abnormalities. One can postulate that the significant lack of agreement may be attributed to the small gestation age of the fetuses and to the retrospective nature of the study. The five cases of fetal MRI interpreted with the presence of cortical abnormalities included the smallest fetuses in the cohort, with gestational ages ranging from 27 to 30 þ 5 weeks. However, the optimal period for MRI imaging to study fetal brain gyration is between 32 and 34 weeks, when extensive cortical sulcation is present. Prior to 30 weeks of gestational age, the evaluation of the simplified sulcation is challenging and remains susceptible to many pitfalls [30]. The symmetry of the images in T2 sequences is of great importance in the evaluation of fetal gyration. In cases of suspected cortical abnormalities, multiple repetitions of the same sequence might be necessary in order to obtain the optimal imaging plane. This ability is not an option in a retrospective study [30].
Subependymal cysts were also quite often identified in neonatal MRIs. Overall, we found high levels of concordance between radiological reports of the same MRI with 90% concordance between fetal MRIs and 100% in neonatal MRIs. Most importantly, a 70% concordance between fetal and neonatal MRIs was noted (Table 2). Finally, although none of the patients presented with symptomatic cCMV infection, 4 out of 10 cases received antiviral therapy at birth, mainly because of their characterization as high-risk patients due to maternal infection taking place early in pregnancy and abnormal fetal and neonatal MRIs.
Our study has several notable limitations. Firstly, since performing both prenatal and postnatal MRIs is not standard practice in children with congenital CMV infection, this was a case series with a small sample size gathered through convenience sampling. Additionally, in many areas of the world no prenatal screening is performed and therefore these results are not easily applicable, since fetal MRIs are performed solely when cCMV is clinically suspected. Therefore, the results of this study are qualitative and cannot be generalized. Secondly, since this was a retrospective study, there was no control over timing of the imaging, therefore we note that both fetal and neonatal MRIs were not performed at the same gestational and postnatal age throughout the cases, respectively. Thirdly, even though there is little evidence on its effectiveness, 9 out of 10 mothers received hyperimmune intravenous immunoglobulin treatment which could have altered the post-natal imaging and outcome. Finally, even though our study was blinded, there was a checklist of CMV related abnormalities created for the radiologists to evaluate, which is not representative of everyday practice.
To our knowledge there is no other study directly comparing the results between fetal and neonatal MRI in children with cCMV infection. Even though this is a small descriptive study, the observations indicate that fetal MRI could potentially provide a diagnostic tool helping clinicians decide when to perform neonatal MRIs in high-risk patients. Of note, our study findings imply that fetal MRIs may overestimate abnormalities with a positive predictive value (PPV) of 70%. Larger prospective studies are needed in order to compare the ability of fetal versus neonatal imaging to identify cerebral abnormalities, as well as to establish the optimal gestational and neonatal age at which it should be performed. Future research should aim to create a universal consensus regarding grading of MRI abnormalities related to cCMV and evaluate its correlation to poor clinical outcome [31]. Over the past few years, it has become evident that we need to move forward from the binary definition of symptomatic versus asymptomatic neonatal cCMV disease and develop a prognostic scale using multiple risk factors associated with adverse outcome, including neuroimaging [32]. Suspicious ultrasound findings during pregnancy, should urge the performance of fetal and neonatal MRI to monitor the evolution of pathological lesions [14]. The combination of PCR testing and neuroimaging will fundamentally aid clinical practice and will assist in the early identification of the asymptomatic neonates that may benefit from prompt antiviral treatment and rigorous follow up.

Disclosure statement
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Funding
The author(s) reported there is no funding associated with the work featured in this article.