(AABR) ACCEPTED. Running title: Perinatal CMV infection and hearing impairment. Leuven, Belgium

Transcription

1 JCM Accepts, published online ahead of print on 3 September 2008 J. Clin. Microbiol. doi: /jcm Copyright 2008, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved. STUDY OF PERINATAL CYTOMEGALOVIRUS INFECTION AND SENSORINEURAL HEARING LOSS IN BELGIAN INFANTS DETECTED BY AUTOMATED AUDITORY BRAINSTEM RESPONSE (AABR) Running title: Perinatal CMV infection and hearing impairment Jannick Verbeeck 1, Erwin Van Kerschaver 2, Elke Wollants 1, Kurt Beuselinck 3, Luc Stappaerts 2, Marc Van Ranst 1* 1 Laboratory of Clinical Virology, Rega Institute for Medical Research, University of Leuven, Leuven, Belgium 2 Kind&Gezin (K&G), Brussels, Belgium 3 Laboratory of Molecular Diagnostics, University Hospital Gasthuisberg, Leuven, Belgium *Corresponding author: Prof. Dr. Marc Van Ranst Laboratory of Clinical Virology, Rega Institute for Medical Research Minderbroedersstraat 10, BE-3000 Leuven, Belgium Tel.: ; Fax: address: marc.vanranst@uz.kuleuven.be Key words: CMV, AABR, hearing impairment, sensorineural hearing loss Word count abstract: 199 Word count article:

2 ABSTRACT Since auditory disability causes serious problems in the development of speech and in the total development of a child, it is crucial to diagnose possible hearing impairment as soon as possible after birth. This study evaluates the neonatal hearing screening program in Flanders, Belgium. The auditory ability of 118,438 babies was tested using the automated auditory brainstem response (AABR). We selected 194 babies with indicative hearing impairment and 332 matched controls to investigate the association between the presence of human cytomegalovirus (HCMV) in urine samples and sensorineural hearing loss (SNHL), and to compare the sensibility and specificity of a cell culture assay and a quantitative PCR detection method. Our results indicate that significantly more babies with confirmed hearing impairment were HCMV positive after birth. Even more, based on the results of our study, babies with HCMV viral loads above 4.5 log copies/ml urine, seem to be 1.4 times more likely to have confirmed hearing impairment. Our follow-up study suggests that the hearing impairment of children infected with HCMV after birth, is less likely to improve compared to HCMV negative infants. Our results confirm that the presence of HCMV before or shortly after birth influences the outcome of hearing impairment. 2

3 INTRODUCTION Human cytomegalovirus (HCMV) is a species-specific member of the β-herpesvirus family. Although HCMV infection in healthy children and adults is usually asymptomatic, it is the leading cause of non-genetic sensorineural hearing loss (a type of hearing loss which is caused by deficits in the vestibulocochlear nerve, in the inner ear or in the central processing centers of the brain) in developed countries (21, 23, 27). Approximately 14% of the infections are diagnosed at birth, while the remaining 86% are asymptomatic (22). HCMV causes mild or subclinical diseases in immunocompromised individuals, such as transplant recipients and AIDS patients (16, 25, 33). The seropositivity rate in the adult population is % (1). This high rate is possibly due to the transmission of the virus through close interpersonal contact, breastfeeding, sexual contact and spread from children. Congenital transmission of HCMV is possible even when maternal immunity is present (19). The incidence of congenitally acquired HCMV in newborns ranges from 0.5% to 2.2% in developed countries (2, 6, 11, 18), but this could be an underestimation since reliable and national-level estimates are not available (14). Up to 22% of neonates with hearing impairment are infected with HCMV (9). The first report on the association between HCMV infection and hearing impairment dates from 1964 (20). Since then, considerable efforts have been made to clarify the exact mechanism of causing hearing loss, but until now, this question has not been resolved yet. Auditory disability has pernicious effects on the development of speech, and on the total development of the child. The age of onset of a child s hearing impairment and the age at which the hearing impairment is diagnosed, are crucial parameters for the further development of the child. Intensive auditive stimulation of the cerebral cortex before the age of six months leads to significant higher language abilities of the disabled children compared to children who do not receive a hearing aid until the age of 7 to 18 months (26, 34). For this reason, universal screening for 3

4 hearing impairment in babies is very important, and this is one of the aims of the Joint Committee on Infant Hearing (JCIH) and the American Academy of Pediatrics. The JCIH guidelines strongly suggest screening before the age of 1 month, with audiological confirmation by 3 months of age in infants who fail the screening test, and initiation of intervention before the age of 6 months (17). The Flanders region in Belgium was the first region in the world that introduced a universal hearing screening program for the whole population (10). Since 1998, more than 90% of the babies born in Flanders are screened for hearing defects by the district nurses from Child & Family, a Flemish public agency whose purpose is to promote welfare and health of all children up to the age of three years (28, 29). The district nurses of Child & Family have contact with virtually all newborn babies via visits at the maternity ward, house calls or consultations at the welfare baby clinic (8). During these visits, every new mother is made aware of hearing impairment screening. At about the age of four weeks, the hearing test (Automated Auditory Brainstem Response or AABR) is carried out by the district nurse. If potential hearing impairment is detected at the first test, a second test is performed within 48 hours in the presence of a baby-clinic medical officer who examines the ears of the child. If the second test is again positive, the baby is immediately referred to a specialized center where the hearing abilities will be further examined. In order to determine the effect of perinatal HCMV infection on hearing impairment, we collected urine samples from newborns with hearing impairment and from healthy controls to examine the presence of HCMV. Children with hearing impairment were enrolled in a followup program for at least two years, in order to assess whether their hearing impairment status changed. 4

5 MATERIALS AND METHODS Study population The auditory ability of 118,438 babies, born between April 2002 and April 2004, was tested with the AABR in two phases. Out of the 194 babies with possible hearing impairment, 45 tested positive for the first test and negative for the second test, and 149 tested positive for both tests. Group 1 contains all babies without confirmed hearing impairment that tested positive for the first test and negative for the second test (n=43), and all babies without confirmed hearing impairment that tested positive for both tests (n=15). In total, group 1 contains 58 subjects. Group 2 consists of all babies with confirmed hearing impairment and a positive result on both tests (n=134) and all babies with confirmed hearing impairment who tested positive for the first test and negative for the second test (n=2). Group 2 contains 136 babies in total. For each baby included in this study, the first 2 babies born in the same region (in a radius of 10 km) and in the same month were matched as controls. Of these, 332 were willing to take part in the study, resulting in a complete dataset of 526 babies (Figure 1). Informed consents were obtained for all children included in this study. Algo hearing screening To evaluate the hearing system of the infants, the AABR or Algo test (Natus Med. Inc., San Carlos, California) was carried out at 4 to 6 weeks after birth at the infant s home. The automated Algo Neonatal Hearing Screener provides a pass-fail report. The apparatus does not require a special test environment or a person who is audiologically trained to interpret the test result. With this apparatus, both ears are automatically tested, and it corrects for ambient noise and for myogenic interference to ensure that data collections are favorable. All tests were carried out in a two-phase screening; babies who tested positive for the first test were conducted to a second test within 48 hours to confirm the result. Normal hearing was defined as thresholds of 35 db at an acoustic frequency spectrum of 700 to 5000 Hz. 5

6 Sample collection and isolation of HCMV from urine samples Urine samples from patients and controls were collected in a time period of 10 to 322 days after birth (average: 49 days). Samples were shipped to the Department of Laboratory Medicine, University Hospital, Leuven where they were immediately stored at -80 upon arrival. Viral DNA was extracted by using the QIAamp DNA Blood Mini Kit (Qiagen Benelux, Leusden, The Netherlands) according to the manufacturer s instructions. Cell culture assay Cell culture assays were immediately performed upon arrival of the samples. Shell vial assays were carried out in embryonic skin muscle cells (E 1 SM) propagated in minimal essential medium 10% (MEM; Gibco, Life Technologies, Rockville, MD, USA) supplemented with 10% fetal calf serum (FCS; Integro, Zaandam, The Netherlands), Mycoplasma Removal Agent (0.1 ml/10 ml MEM 10%), and 0.5% sodium bicarbonate (Gibco). The cell suspension was maintained at 37 C in a humidified 5% CO 2 atmosphere. Before inoculation, MEM was removed from the shell vial, and 0.5 ml urine was added directly to the cell monolayer. The shell vials were closed and centrifuged at 1850 rpm at 25 C for 1h. After viral adsorption and centrifugation, the urine specimen was removed from each vial, and 2 ml MEM supplemented with 2% FCS was added to each vial. The cultures were incubated at 37 C in a humidified 5% CO 2 atmosphere for 24 h. Cell monolayers were stained by using the monoclonal anti-hcmv Immediate Early Antigen (I.E.A.; Argene, Biosoft, LabConsult SA, Brussels, Belgium) according to the manufacturer s instructions. HCMV positive cells were counted by visual screening with a fluorescence microscope. Quantitative PCR assay A quantitative PCR was designed in the major capsid gene region of the HCMV genome, amplifying a 340 bp fragment (5). Briefly, a 50 µl TaqMan PCR was carried out using 10 µl of extracted DNA, 20 µl of Universal Mastermix 2X containing ROX as a passive reference 6

7 (Applied Biosystems), 0.25 µm forward primer 5 -CGTAACGTGGACCTGACGTTT-3, 0.25 µm reverse primer 5 -CACGGTCCCGGTTTAGCA-3, and 0.20 µm probe 5 - TATCTGCCCGAGGATCGCGGTTACA-3, which was labeled at the 5 end with the fluorescent dye 6-carboxyfluorescein (FAM) as the reporter dye, and the 3 end was labeled with quencher dye 6-carboxytetramethylrhodamine (TAMRA) (Eurogentec, Seraing, Belgium). Amplification and detection were performed using a ABI PRISM 7700 Sequence Detection System (Applied Biosystems) under the following conditions: an initial annealing step for 2 min at 50 C followed by a PCR activation step at 95 C for 10 min and 45 cycles of amplification (15 sec at 95 C and 1 min at 60 C). During amplification, the ABI PRISM sequence detector monitored real-time PCR amplification by quantitative analysis of fluorescence emissions. The reporter dye (FAM) signal was measured against the internal reference dye (ROX) signal to normalize for non-pcr-related fluorescence emissions. The threshold cycle was defined as the fractional cycle number at which the reporter fluorescence, generated by cleavage of the probe, reaches a threshold defined as 10 times the standard deviation of the mean baseline emission between cycle 3 and 15. Statistical analysis All statistical tests were carried out using SPSS version The significance level was set at P<

8 RESULTS Statistical description of dataset Between April 2002 and April 2004, a total of 128,765 babies were born in the Flemish region of Belgium. Of these, 3,497 were born on the Neonatal Intensive Care Unit, and 925 babies died during the first 6 weeks after birth. Due to the network of Child & Family, 92% of the babies born in this period (i.e. 118,438 babies) were screened for possible hearing impairment using the AABR or Algo test in a two-phase screening. Group 1 consists of 58 babies without confirmed hearing impairment who tested positive for both hearing test or with an inconclusive result. Group 2 comprises 136 babies who have confirmed hearing impairment with an inconclusive result or with positive results on both hearing tests. In total, 332 controls were matched (Figure 1). Hearing impairment at birth was confirmed by an audiological centre in 136 (25.9%) out of the 526 tested babies (all babies in group 2), while 390 (74.1%) tested babies proved to have normal hearing. A urine sample was collected from all babies, and a cell culture assay for the detection of HCMV was carried out on these samples. The average time between the date of birth of the baby and the sample testing date was 49 days (42 days [range: 10 to 125 days] for controls, 67 days [range: 14 to 266 days] for group 1, and 57 days [range: 13 to 322 days] for group 2). By using the cell culture assay, a total of 29 babies (5.5%) tested positive for the presence of HCMV, of which 11 babies belonged to the control group, 4 to group 1 and 14 to group (93.3%) babies tested negative (317 controls, 54 in group 1 and 120 in group 2). For 6 children (1.1%), the result of the cell culture assay was undefined (4 controls and 2 in group 2). Sensitivity of quantitative PCR assay A quantitative PCR was designed in order to search for false negative or false positive results (5). All samples from patients and controls were analyzed in duplicate and the same positive 8

9 control was used for each run. A no template control (where H 2 O was added instead of extracted DNA) was used as negative control. The quantitation standard curve was achieved by using 10-fold serial dilutions of HCMV DNA, corresponding to copy numbers per ml of 5 x 10 2 to 5 x The slope of the obtained standard curve was -3.36, and the Y-intercept (i.e. the theoretical Ct value to detect one copy) was With these values, the following formula could be generated for quantitation of HCMV DNA in the samples: log HCMV copies/ml = (Ct 45.7)/ Viral loads between 1.86 log copies/ml and 7.48 log copies/ml were detected. By using this quantitative PCR, 489 urine samples tested negative for the presence of HCMV, and 37 samples were positive. The samples that were undefined based on the cell culture assay results, could now be unambiguously interpreted (Table 1). Urine samples that were found positive with our quantitative PCR, are defined as true positives. Association between the presence of HCMV and confirmed hearing impairment A possible association between the presence of HCMV and confirmed hearing impairment for the different groups was examined statistically. Table 2 shows the number of infants in each group, according to the presence or absence of HCMV in their urine. Based on the results of both the cell culture assay and the quantitative PCR, the number of infants in group 2 that are HCMV positive (10.3% for cell culture assay and 12.5% for quantitative PCR) is three times higher compared to the HCMV positive samples in the control group (3.3% for cell culture assay and 4.5% for quantitative PCR). A Pearson Chi-Square test indicated that this difference was statistically significant (χ 2 =9.354; p=0.002) for the results of the cell culture assay. There was no significant difference between the babies of group 1 and group 2 (χ 2 =0.601; p=0.44), and between the control group and group 1 (Fisher s Exact test, p=0.257), although there are approximately two times more HCMV infections in group 1 compared to the control group. The same statistical analysis was repeated with the results of the 9

10 quantitative assay. Again, the Pearson Chi-square test indicated that more babies tested positive in group 2 compared to the control group (χ 2 =9.649; p=0.002). No significant difference could be detected between controls and infants in group 1 (Fisher s Exact test, p=0.197), or between infants in group 1 and infants in group 2 (χ 2 =0.609; p=0.43). To assure that this significant result was not an artifact caused by a possible difference in delay of sample arrival between the controls and group 2, we calculated the average time between the date of birth and the date of sample arrival for both groups (42 days [range: 10 to 125 days] for controls versus 57 days [range: 13 to 322 days] for group 2). A non-parametric Mann-Whitney test showed that the average time for group 2 was significantly higher (p=0.0001) in comparison to the control group, indicating that urine samples of infants in the control group were collected and analyzed sooner after birth. This finding also implicates that for babies in group 2, there was a significant longer perinatal time period in which they could be infected with HCMV. HCMV viral load and confirmed hearing impairment We also examined a possible relationship between viral load and confirmed hearing impairment. Figure 2 shows the percentage of children with congenital HCMV infection and assigned hearing loss according to the virus quantity in the urine samples. Almost 60% of the infants with confirmed hearing loss had viral loads of at least 4.5 log copies/ml, suggesting a positive correlation between viral load and hearing impairment. The odd s ratio is (95% CI: ), and children with a viral load of 4.5 log copies/ml or higher, are 1.4 times more likely to have confirmed hearing impairment than children with lower viral loads. Follow-up of babies with confirmed hearing impairment All infants with confirmed hearing loss soon after birth, were invited to participate in a follow-up study to evaluate their hearing abilities after 24 months. After this time, data of 70 infants could be collected. We were most interested in children with unilateral hearing loss 10

11 who were HCMV positive, because they are at high risk for developing bilateral hearing loss. Table 3 shows the number of infants with unilateral or bilateral hearing loss (determined at birth and after 24 months), according to the presence or absence of HCMV at birth or soon after birth. Of the four children, all four HCMV positive, with unilateral hearing impairment at birth, 1 developed bilateral hearing loss after 24 months. Of the HCMV positive children with bilateral hearing loss at birth, none showed hearing improvement during the follow-up period. In contrast, 3 children with bilateral hearing loss at birth who were HCMV negative, evolved to a unilateral hearing loss status after 24 months. Nine HCMV negative children with bilateral hearing loss at birth, and 6 with unilateral hearing loss, did not show hearing impairment after 24 months. None of the children had developed delayed onset hearing loss at the end of the study. 11

12 DISCUSSION Up until 1997, detection of hearing deficits in infants was performed with the Ewing test (behavioral test) at the age of approximately 9 months. Revalidation procedures for infants with partial or total hearing loss started at the age of 2 years, which is too late for requiring optimal results. Since 1998, hearing screening is performed at the age of 4 to 6 weeks with the AABR, and integral revalidation of the infants starts at the age of 3 months. The Flanders region in Belgium was the first geographical region where hearing screening was offered to virtually all newborns. The Child & Family public agency succeeds in screening more than 90% of all newborns in Flanders every year, which is a high percentage in comparison with the Early Hearing Detection and Intervention Programs (EHDI) in America, where 70% of all infants born are screened (31). Moreover, there is almost no loss of follow-up of referred children with hearing impairment in Belgium. The decision to set up a screening campaign is based on a comparison of the expected benefits and costs of screening and treatment. Moreover, effective and productive screening involves an assessment of the importance of the disease as a public health problem. Neonatal screening for hearing impairment meets all these criteria, and is therefore accepted in most countries (4). Most countries choose to perform the neonatal hearing screening before discharge out of the maternity unit. However, different studies have shown that testing soon after birth results in a greater percentage of false positive results (24, 30), which undermine the credibility of the test, and lead to unnecessary pressure for parents and personnel. Also from a therapeutic point of view, there is no reason for testing infants directly after birth, since there are no immediate solutions for hearing improvement, and revalidation is only offered from the second or third month after birth. For these reasons, the AABR in Flanders is conducted at the age of 4 to 6 weeks. At this age, the average AABR test time is the shortest, which is another advantage of this policy. 12

13 Since it is essential to diagnose perinatal HCMV infection with a specific, sensitive and rapid method, we evaluated a quantitative PCR to detect HCMV in urine samples, and we compared this method with the shell vial assay which is considered as the gold standard method for virus isolation from urine. Our results indicate that the quantitative PCR is a sensitive and reliable alternative for the cell culture assay. Moreover, results that were uninterpretable with the shell vial assay, could unambiguously be interpreted with the quantitative PCR. In our dataset, 7% (37 out of 526) of the babies tested positive for the presence of HCMV with the quantitative TaqMan assay, of which 38% (14 out of 37) belonged to group 2. This result indicates that HCMV infected newborns seem to have a higher risk to develop hearing impairment soon after birth compared to HCMV negative babies and that the AABR screening test is a reliable method to identify possible hearing problems in newborns. In total, 12.5% (17/136) of the babies with confirmed hearing impairment were infected with HCMV, and this number equals the numbers found in other studies on HCMV-related sensorineural hearing loss (7, 12, 15, 32). Of the 17 HCMV positive babies with confirmed hearing impairment, 10 had viral loads above 4.5 log copies/ml urine. This means that babies with a high viral load of at least 4.5 log copies seem to be 1.4 times more likely to have confirmed hearing impairment, although further research with larger datasets is necessary to confirm this finding. Our results indicate that higher viral loads correlate with a higher likelihood of hearing loss. Unfortunately, we did not have additional information of all babies, such as birth weight or other risk factors for HCMV infection, so we could not include these factors in our statistical analysis. Nevertheless, CMV viral load determination in the urine of newborns could be an important complement to hearing screening for the early detection of possible hearing loss and should therefore be validated as a possible predictive factor for the risk of development of hearing impairment (14). 13

14 Since the screening for HCMV was not performed on Guthrie cards or umbilical cord samples, but on urine samples that were collected on average 49 days after birth, we can not be sure that the HCMV positive children were congenitally infected. The incidence of congenitally acquired HCMV infections is 0.4% to 2.2% (2, 6), and these number are lower than the number of HCMV positive children in our dataset, which suggests that most infections were probably acquired perinatally. Sensorineural hearing loss is not always present at birth, and can begin months or even years after birth (13, 24). Fowler and colleagues showed that the most important decline in hearing occurs during the first year of life (12). For this reason, intensive follow-up of children at risk for developing hearing impairment is very important. In this study, we evaluated the hearing abilities of 70 children after 24 months, and studied the long term differences between children who were HCMV positive and HCMV negative at birth. The results of our follow-up study suggest that children with HCMV-related hearing impairment experience a continued deterioration of their hearing abilities during childhood, which confirms previous findings (3, 7, 13, 32). Hearing impairment in children who were not infected with HCMV at birth seems to be less profound and has a more temporary nature. These findings could be important in therapeutic interventions, but further research has to be undertaken to study possible spontaneous amelioration of hearing impairment. Since early detection of hearing impairment in infants is beneficial for the development of their language skills and for their educational outcome, universal screening for congenital and perinatal HCMV infection could be an important tool to identify children who will profit from intensive audiological follow-up. 14

15 ACKNOWLEDGMENT This work was supported by the Flemish Fund for Scientific Research (Fonds voor Wetenschappelijk Onderzoek, FWO) grant G We would like to thank Prof. Dr. F. Eyskens of the Provinciaal Centrum voor Opsporing van Metabole Aandoeningen, Antwerp, Prof. Dr. B. Wuyts of the University Hospital, Ghent, and Prof. Dr. D. Bernard of the Centrum voor Opsporing van Aangeboren Metabole Aandoeningen, Bruges, for their help. We also thank the nurses of Child & Family, Brussels for collecting the urine samples, and our colleagues at the Department of Laboratory Medicine, University Hospital, Leuven, for technical support. 15

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18 18. Kenneson, A., and M.J. Cannon Review and meta-analysis of the epidemiology of congenital cytomegalovirus (CMV) infection. Rev. Med. Virol. 17: Kumar, M.L., G.A. Nankervis, I.B. Jacobs, C.B. Ernhart, C.E. Glasson, P.M. McMillan, and E. Gold Congenital and postnatally acquired cytomegalovirus infections: long-term follow-up. J. Pediatr. 104: Medearis, D.N Observations concerning human cytomegalovirus infection and disease. Bull. Johns Hopkins Hosp. 114: Mocarski, E.S., and C.T. Courcelle Cytomegaloviruses and their replication, p In D.M. Knipe, P.M. Howley, et al (ed). Fields Virology. 4 th edition. Vol 2. Philadelphia: Lippincott Williams & Wilkins. 22. Nance, W.E., B.G. Lim, and K.M. Dodson Importance of congenital cytomegalovirus infections as a cause for pre-lingual hearing loss. J. Clin. Virol. 35: Pass, R.F Cytomegalovirus, p In D.M. Knipe, P.M. Howley, et al (ed). Field Virology. 4 th edition. Vol 2. Philadelphia: Lippincott Williams & Wilkins. 24. Peckham, C.S., O. Stark, J.A. Dudgeon, J.A. Martin, and G. Hawkins Congenital cytomegalovirus infection: a cause of sensorineural hearing loss. Arch.Dis. Child. 62: Rubin, R.H., N.E. Tolkoff-Rubin, D. Oliver, T.R. Rota, J. Hamilton, R.F. Betts, R.F. Pass, W. Hillis, W. Szmuness, M.L. Farrel, and M.S. Hirsch Multicenter seroepidemiologic study of the impact of cytomegalovirus infection on renal transplantation. Transplantation 40: Sokol, J., and M. Hyde Hearing screening. Pediatr. Rev. 23:

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20 TABLES Table 1: Distribution of the samples in the different groups, according to the results of the cell culture assay and the quantitative PCR assay. Negative quantitative PCR Negative cell culture Positive cell culture Undefined cell culture Negative cell culture Positive quantitative PCR Positive cell culture Undefined cell culture Group Total Group Controls Total Table 2: Number (percentage) of babies in the three different groups according to the presence or absence of HCMV, as defined by the cell culture assay and the quantitative PCR. HCMV positive HCMV negative Cell culture Quantitative PCR Cell culture Quantitative PCR Group 1 (%) 4 (6.9%) 5 (8.6%) 54 (93.1%) 53 (91.4%) Group 2 (%) 14 (10.3%) 17 (12.5%) 120 (88.2%) 117 (86.1%) Controls (%) 11 (3.3%) 15 (4.5%) 317 (95.5%) 317 (95.5%) Table 3: Number of infants with unilateral and bilateral hearing loss, for whom results were available at birth and after a 24 month follow-up, according to the presence of HCMV as determined by the quantitative PCR. Assigned hearing loss at birth Assigned hearing loss after 24 months 20

21 Unilateral Bilateral Unilateral Bilateral None HCMV pos HCMV neg Total

22 FIGURE LEGENDS Figure 1: Schematic overview of the study population. Figure 2: Viral loads (log copies/ml urine) in HCMV positive urine samples for children with confirmed hearing impairment (white bars) and controls (black bars) defined by using the quantitative PCR. 22

23 FIGURE 1 118,438 babies screened for possible hearing impairment Pass result Pool for control group: 332 healthy controls defined as the first two babies born in the same region and month as the 194 Refer babies first AABR test Group 1 (58 babies) Pass or Refer result without confirmed hearing impairment Refer result second AABR test Group 2 (136 babies) Pass or Refer result with confirmed hearing impairment

24 FIGURE 2 Percentage of children ,5 46,7 33,3 58,8 17,6 20 <3,0 3,0-4,5 >4,5 Viral load (log copies/ml)