The Pediatric Infectious Disease Journal Publish Ahead of Print

DOI: 10.1097/INF.0000000000001630

Newborn Congenital Cytomegalovirus Screening based on Clinical Manifestations and

Evaluation of DNA-based Assays for In Vitro Diagnostics

Tomoyuki Fujii, MD, PhD1, Akira Oka, MD, PhD2, Ichiro Morioka, MD, PhD3, Hiroyuki

Moriuchi, MD, PhD4, Shin Koyano, MD, PhD5, Hideto Yamada, MD, PhD6, Shigeru Saito,

MD, PhD7, Hiroshi Sameshima, MD, PhD8, Takeshi Nagamatsu, MD, PhD1, Shinya Tsuchida,

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MD, PhD2, and Naoki Inoue, PhD9*, for the Japanese Congenital Cytomegalovirus Study

Group #

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Departments of 1 Obstetrics and Gynecology and 2 Paediatrics, The University of Tokyo,

Tokyo; Departments of 3 Paediatrics and 6 Obstetrics and Gynecology, Kobe University,

Hyogo; 4 Department of Paediatrics, Nagasaki University, Nagasaki; 5 Department of

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Paediatrics, Asahikawa Medical University, Hokkaido;7 Department of Obstetrics and

Gynecology, University of Toyama, Toyama; 8 Department of Obstetrics and Gynecology,

University of Miyazaki, Miyazaki, and 9 Department of Microbiology and Immunology, Gifu

Pharmaceutical University, Gifu, JAPAN

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#
Members of the Japanese Congenital Cytomegalovirus Study Group who contributed to this

study are listed in the Acknowledgement section.

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* Corresponding author: Naoki Inoue, Department of Microbiology and Immunology, Gifu

Pharmaceutical University, 1-25-4 Daigaku-Nishi, Gifu 501-1196, JAPAN

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+81-58-230-8125 (Phone/FAX), inoue@gifu-pu.ac.jp (e-mail)

Abbreviated title: Risk-Based Newborn Screening and In Vitro Diagnostics for Congenital

CMV

Running head: Screening Approach & In Vitro Diagnostics for Congenital CMV

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 COI and Funding support: None of the authors has any conflict of interest to declare. This

work was supported by a Grant for the Research on Child Development and Diseases

(15gk0110003h0103) from the Agency for Medical Research and Development (AMED), the

Japanese Government.

ACKNOWLEDGMENTS

We would like to thank our medical and nursing colleagues as well as the newborns and their

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parents who agreed to take part in this study. We would like to also thank the following

members of the Japanese Congenital Cytomegalovirus Study Group for their contributions: H.

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Samejima (Samejima Bonding Maternity Clinic, Saitama), S. Yamaguchi (Yamaguchi

Hospital, Chiba), K. Nishida, T. Koda, S. Iwatani (Kobe Univ.), M. Ishibashi (Nagasaki

Univ.), M. Itoh (Univ. Toyama), Y. Kawagoe (Univ. Miyazaki). All individuals listed are part

of the Study Group and have not received any compensation.

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 ABSTRACT

Objectives: To establish a strategy for congenital cytomegalovirus (cCMV) screening and to

establish confirmatory assays approved as In Vitro diagnostics (IVD) by the regulatory

authorities, we evaluated the clinical risks and performance of diagnostic assays developed by

commercial companies, since cCMV infection has significant clinical consequences.

Study design: Newborns with clinical manifestations considered to be consequences of

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cCMV infection (n=575) were screened for the presence of CMV DNA in urine specimens

collected onto filter paper placed in their diapers using the PCR-based assay reported

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previously. Liquid urine specimens were obtained from all of 20 CMV-positive newborns

and 107 of the CMV-negative newborns identified in the screening. We used these 127

specimens, as well as 12 from cCMV cases identified in a previous study and 41 from healthy

newborns, to compare the performance of two commercial assays and one in-house assay.
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Results: The risk-based screening allowed the identification of cCMV cases at least 10-fold

more efficiently than our previous universal screening, although there appears to be a limit to

the identification of asymptomatically-infected newborns. Although CMV-specific IgM

during pregnancy was found frequently in mothers of cCMV newborns, CMV-IgM alone is
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not an effective diagnostic marker. The urine-filter based assay and the three diagnostic

assays yielded identical results.

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Conclusions: Although risk-based and universal newborn screening strategies for cCMV

infection each have their respective advantages and disadvantages, urine-filter based assay
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followed by confirmatory IVD assays is able to identify cCMV cases efficiently.

Keywords: clinical manifestations, congenital cytomegalovirus infection, in vitro diagnostics,

newborn screening

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 INTRODUCTION

Congenital cytomegalovirus (cCMV) infection occurs in 0.2-2% of births in developed

countries and is associated with significant clinical consequences, not only at birth but also

later as neurological sequelae, including sensorineural hearing loss (SNHL) and

developmental delays 1. Our retrospective studies demonstrated that fetal cCMV infection

was associated with in 12-15% of cases of severe SNHL and 25% of cases of developmental

delay of unknown cause, with one half of the sequelae being late-onset 2,3. Early

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identification of cCMV infection may lead to new treatment options with antiviral agents 4

and early intervention in infants with SNHL to aid language development 5. Therefore, it is

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important to establish newborn cCMV screening programs.

Traditionally, the diagnosis of cCMV infection has been performed by CMV isolation

from urine specimens collected within 3 weeks of birth. As the virus isolation is laborious,
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PCR detection of CMV DNA in urine specimens is used as the gold standard assay. However,

collection of urine specimens in liquid form is tedious, so we developed a quantitative PCR

(qPCR) assay using urine specimens collected on filter discs (urine-filter) that can be used

directly as a PCR template without additional purification or elution steps 6,7, and
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demonstrated the feasibility of our assay for relatively large-scale screening programs 810. To

implement universal screening programs, it is also essential to establish confirmatory assays

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recognized as In-Vitro diagnostics by the regulatory authorities.

In this study, we conducted screening based on clinical manifestations considered to be

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risk factors for cCMV infection, and evaluated the significance of each risk factor in the

identification of cCMV newborns. In addition, to establish confirmatory assays that can be

recognized as In Vitro diagnostics, we evaluated the clinical performance of diagnostic

reagents developed by two commercial companies using urine specimens obtained from the

newborns enrolled in the screening described above.

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 MATERIALS and METHODS

Study design

As shown in Fig. 1, this study consisted of two parts. Part A sought to establish the

significance and limitations of newborn CMV screening based on clinical manifestations and

features indicative of cCMV at five study sites (Tokyo, Kobe, Nagasaki, Toyama, and

Miyazaki) from Oct. 1, 2014 to Nov. 30, 2015. Five hundred and seventy-five newborns who

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exhibited at least one of the clinical manifestations and serological indications listed in Table

2, including fetal and neonatal symptoms as well as maternal features of possible CMV

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infection during pregnancy, were enrolled for CMV screening using the urine-filter based

qPCR assay.

Part B sought to evaluate the clinical performance of assays developed by commercial

companies (Shino-Test Corporation and QIAGEN Japan), and the results of the study were
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intended to be used in the application of these two commercial assays as In-Vitro Diagnostics

recognized by the regulatory authorities. The minimum numbers of CMV-positive and

negative specimens for enrollment of the Part B study were determined based on suggestions

by the regulatory authorities ahead of the study. Urine specimens in a liquid form were
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collected both from all CMV-positive newborns (n=20) and a portion of the CMV-negative

newborns identified during screening at two of the study sites (n=107) as well as from a
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number of healthy newborns (n=41). The selection of the CMV-negative newborns was done

randomly from the two study sites so as to make the numbers almost equal between the sites
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(n=53 in the Tokyo area, and n=54 in the Kobe area). For the selection, the Excel RAND

function was used. Collection and use of the human subject materials was approved by the

Ethical Committee on Human Subjects of each participating institute and of the two

companies. Informed consent from the parents of the respective newborns was obtained at

enrollment for collection of liquid urine specimens for evaluation of the assays developed by

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 the two companies. All specimens collected for screening and evaluation of the assays were

anonymized and only the attending physicians can link the assay results to their patients. In

addition, the original code numbers were randomized and recoded to ensure that the assay

performance evaluation was conducted in a completely blind manner.

Clinical evaluation

Clinical information for the newborns, including birth weight, gestation age, clinical

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manifestations, and abnormal laboratory findings, were extracted from their medical records.

Audiological testing was performed using auditory brainstem responses and/or auditory

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steady-state responses, and brain imaging was performed by echography, computed

tomography and/or magnetic resonance imaging. The maternal medical records were

examined for abnormalities during pregnancy. Serologic tests for CMV-specific IgG and IgM

for the pregnant mothers were performed at a commercial laboratory (SRL Inc., Japan) using
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EIA kits produced by DENKA SEIKEN Co. Ltd (Niigata, Japan). The CMV-IgM kit is based

on the IgM-captured sandwich method using CMV-specific antibodies for detection.

qPCR for screening

Urine samples were collected from newborns onto filter paper within 1 week after birth, and
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the presence of CMV-DNA was assessed as described previously 7 with minor modifications,

including the use of No.4A filter paper (ADVANTEC, Tokyo, Japan) instead of FTA-Elute
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Card and removal of the procedure for washing the filter discs with water prior to qPCR

reaction. These modifications allowed for reduction in cost, potential for minor skin
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irritations (change in filter paper) and labor (removal of the washing procedure). qPCR

assays with filter paper spiked with CMV-positive or -negative urine specimens validated that

the modifications did not affect assay performance.

DNA-based assays

The in-house real-time qPCR assay using liquid urine samples was performed as described

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 previously 2. This was one of the assays used for the collaborative study to evaluate the

proposed 1st WHO International Standard for CMV. The assay produced by QIAGEN was

also based on a qPCR assay, and used reagents included in the artus CMV RGQ MDx kit,

which is approved for monitoring CMV viral loads in transplant patients in the US. Two

hundred copies per reaction was used as cut-off for the positive detection of CMV DNA in

this study. The assay developed by Shino-Test Corp. was based on the asymmetric isothermal

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amplification method, known as the Smart Amplification Process (SMAP), originally

developed by Riken 11, and its conditions were similar to those described previously for CMV

detection 12 apart from the use of a different set of primers (IVD application submission no.

DNA.

Statistical analyses TE
5122858000105). Incubation for 60 min was used as a cut-off point for the detection of CMV
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Statistical significance in the prevalence of cCMV between study areas was evaluated using

the Chi-square test. Correlations between viral copy numbers obtained by two different

methods were evaluated by Pearsons correlation coefficient.

RESULTS
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Significance of clinical manifestation-based screening

Screening of 575 newborns for cCMV infection based on the clinical manifestations listed in
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Table 2 as risk factors identified 20 newborns with cCMV infection across the 5 study sites.

As 3 of the 5 study sites participated in the previous study based on the universal screening of
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newborns for cCMV infection 8, we compared the detection frequencies of cCMV infection

between the risk-based and universal screening approaches. As shown in Table 1, the risk-

based screening provided an at least 10-fold increase in the detection of newborns with

cCMV. Although there was ~2-fold difference in the prevalence of cCMV infection between

study sites, the difference was not statistically significant.

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 Association between clinical manifestations and cCMV infection

We obtained clinical information for the 20 CMV-positive newborns as well as for 107 CMV-

negative infants, which corresponded to about one-fourth of the total population of CMV-

negative newborns identified during the urine-filter-based screening at the Tokyo and Kobe

study sites (Table 2, manifestations of each patient are shown in on-line only Table,

Supplemental Digital Content 1, http://links.lww.com/INF/C726). The ratio of cases of

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symptomatic cCMV, defined as those with typical clinical manifestations (hearing

impairment, chorioretinitis, microcephaly, hydrocephalus, or petechiae/ecchymosis) and/or

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with the abnormalities in brain images, was 60%, with 14 of the 20 CMV-positive newborns

exhibiting at least one of the clinical manifestations (average 4.1 manifestations; excluding

maternal indications). When compared with the clinical manifestations of the 107 CMV-

negative newborns, representing common symptoms observed in neonates, the characteristic

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features of cCMV seemed to be a tendency toward bleeding and CNS features, such as

hearing impairment, microcephaly, hydrocephalus, and intracerebral calcification. Although

more than one-third of the cCMV cases exhibited intrauterine growth restriction, low birth

weight, and jaundice that required treatment, these manifestations were also identified in the
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CMV-negative newborns at similar frequencies.

As 50 of 96 among the 107 CMV-negative newborns and 10 of the 13 cCMV newborns

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had CMV-IgM-positive mothers, it is estimated that around 5% of the CMV-IgM-positive

mothers across the 5 study sites delivered cCMV newborns (see the equation in the Footnote 1
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at the end of text). Among the 50 CMV-negative and 10 CMV-positive newborns from

CMV-IgM positive mothers, 9 and 6 of them, respectively, had additional indications for

cCMV infection (Table 3). Due to the limited number of specimens, it is hard to predict any

relationship of CMV-IgM-positivity with a particular manifestation.

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 Comparison of the DNA-based assays for cCMV infection

Urine specimens in liquid form were collected from 180 newborns (Fig. 1) and used for the

performance evaluation of the assays developed by two commercial companies. Urine

specimens from 41 healthy newborns and from 107 newborns with clinical manifestations

who were found to be CMV-negative in the screening assay were all CMV-negative, and

those from the 20 newborns with clinical manifestations who were found to be CMV-positive

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in the screening assay and from 12 CMV-positive newborns identified in the previous study

were all CMV-positive in all of the three assays; i.e., the qPCR assay developed by QIAGEN,

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the SMAP-based assay by Shino-Test, and our in-house qPCR assay.

This study did not focus on the determination of the quantitative correlation among the

assays as the DNA-based assays developed by the two companies are intended to clinically

diagnose the presence or absence of congenital CMV infection. However, to identify any
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potential limitations in each assay, quantitative measurements by the three assays were

compared. As shown in Fig. 2A, there was a good correlation for the in-house and QIAGEN

qPCR assays (r=0.93). In contrast, the in-house qPCR and the Shino-Test SMAP assay

showed a weaker correlation (r=0.66) (Fig. 2B). There was one obvious outlier (case 71),
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marked with an arrow, and exclusion of this outlier resulted in a better correlation (r=0.86).

As the target sequence region for the SMAP assay of the outlier contained one base alteration
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in the detection primer sequence, it is plausible that the alteration decreased the detection

efficiency of the SMAP reaction. Further, as the sequences for the primers in the target region
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of the SMAP assay were well conserved among 19 Japanese clinical isolates and 4 laboratory

strains 13, the base alteration identified in this study appears to be very rare.

DISCUSSION

There were three major outcomes of our study. First, CMV screening based on clinical

manifestations was able to improve the efficiency of CMV screening by at least 10-fold,

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 although one-third of cCMV cases may be overlooked as discussed below. Second, as the

DNA-based CMV assays developed by two companies and our in-house qPCR assays

provided consistent results, both the commercial assays are considered to be reliable for use as

an In Vitro diagnostics for the determination of cCMV infection in newborns. Finally, the

urine-filter-based assay is a reliable tool for newborn CMV screening, with the 107 CMV-

negative and 20 CMV-positive results obtained in the screening tests of newborns with

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clinical manifestations being completely consistent with those obtained by the 3 other

confirmatory assays.

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We previously reported that i) typical clinical manifestations, defined as any of

microcephaly, chorioretinitis, SNHL, or a combination of petechiae, hepatosplenomegaly, and

jaundice, were observed in 22.7% (15/66) of the infected newborns, while ii) abnormalities in

brain images, including intracranial calcifications, ventricular dilation, and abnormal lesions,
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were identified in 10 out of 58 cases, so that iii) the proportion of symptomatic cases with

typical clinical manifestations and/or abnormalities in brain images was 30.3 % (20/66) 8.

The proportion of symptomatic cCMV cases in this study was two-fold (60%) that in the

previous study, while the risk-based selection allowed the at least 10-fold more efficient
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identification of cCMV cases. These findings suggest that a risk-based screening approach is

feasible in situation in which there are insufficient resources for the introduction of universal
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screening. The ~2-fold difference in the prevalence of cCMV infections between the study

sites, although it was not statistically significant, could be due to the difference in the ratio of
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type-1 study sites (primary obstetric clinics and municipal hospitals) against type-2 sites

(university-associated and government hospitals) between the study areas as discussed in our

previous publication 8.

However, there is an obvious drawback in the risk-based approach; that is, the potential

to overlook asymptomatic cases. Among the 66 cCMV cases in the previous study, 10 cases

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 presented with one or two non-specific manifestations, including low birth weight (n=8),

jaundice (n=2), and mild liver function abnormality (n=2), in addition to the 20 cases with

symptomatic cCMV. Thus, 45.5% (30/66) of cCMV cases could be identified by clinical

manifestations observable in the fetuses and newborns. This study found that the detection of

CMV IgM or seroconversion in the mothers, in the absence of any other clinical

manifestations, could identify 30% (6/20) of cCMV cases. Taken together, a simple

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calculation based on this information shows that the screening approach based on a

combination of clinical manifestations and serology may be able to identify about two-thirds

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(see the equation in the Footnote 2 at the end of text) of cCMV cases; in other words, such an

approach may overlook one-third of cCMV cases, particularly asymptomatic cases or those

with milder signs at birth.

The clinical manifestations used for the screening were selected based on previously
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reported observational studies 1419. Among them, thrombocytopenia, petechiae and

hydrocephalus were the most useful predictors for cCMV infection. SNHL is the most

common feature of cCMV infection, and its early detection and subsequent therapeutic

intervention using antiviral agents remain important clinical issues. Although universal
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neonatal hearing screening is helpful, screening coverage is limited to approximately two-

thirds of all newborns in Japan. In addition, late-onset SNHL is well known to be associated
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with the clinical course of cCMV, and the DNA-based diagnosis of cCMV infection during

neonatal period is crucial. Examination of brain images during the early phase of infancy are
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not routinely indicated in the absence of other clinical manifestations. Actually, all of the 7

newborns with SNHL and 6 with brain image abnormalities had at least 2 additional

manifestations. Therefore, most of newborns with SNHL or brain image abnormalities could

be selected and subjected to the screening test based on apparent clinical signs, leading the

correct diagnosis. The finding of a small positive predictive value for CMV-IgM positivity in

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 pregnant mothers for cCMV infection is consistent with the results of our previous study 20.

However, in terms of cCMV screening, information on maternal CMV-IgM status would

provide a useful way to increase the detection of cCMV cases in the population. The

introduction and standardization of serologic assays for CMV-IgG avidity is likely to increase

the efficacy of CMV serology. Nevertheless, it is well documented that cCMV infection can

be caused not only primary infection but also reinfection and reactivation 2124.

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As CMV-specific antigenemia assay and IgM serology using umbilical or neonatal blood

specimens can detect only a portion of newborns with cCMV infection 8, and since CMV

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DNA copy numbers in urine specimens are significantly higher than those in blood specimens

from newborns with cCMV infection 6, DNA-based assays using urine specimens are

expected to perform better than any other assays. Although saliva specimens contain the

amount of CMV DNA similar to urine specimens, there is a potential for contamination with
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CMV DNA from breast milk 25. The performance of all three assays in the diagnosis of

cCMV was adequate as expected. However, the quantitative analysis of the performance of

the assays revealed that some urine specimens had lower copy numbers of CMV DNA. As

those specimens were mainly from the previous study and had shown high copy numbers
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immediately after collection, it is considered likely that the freeze-thawing of the urine

materials a couple of times for use in other studies degraded the DNA.
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In conclusion, the screening and confirmation of cCMV infection by the urine-filter-

based assay followed by either of the two commercial assays as an In Vitro diagnostics
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affords appropriate identification of cCMV infection in newborns, although the risk-based and

universal screening approaches may show differences in efficiency and comprehensiveness in

the of identification of cCMV in newborns.

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 FOOTNOTES

1. 1) The CMV-IgM-positive frequencies

CMV-negative newborns 49/96 and cCMV newborns 10/13

2) 20 cCMV newborns were identified from 575 newborns (20 CMV+, 555 CMV -)

Thus, (20x10/13)/(20x10/13+555x50/96)=0.051

2. Among the 66 cCMV cases identified by the universal screening program in the previous

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study,

1) Cases whose cCMV could be identified by clinical manifestations =30

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2) Cases w/o clinical manifestations but whose cCMV could be identified only by

serology=(66-30)x30%=10.6

Thus, (33+10.6)/66 = 62% of cCMV cases in the previous study could be presumably

identified.
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 FIGURE LEGENDS

Figure 1. Study design and numbers of newborns enrolled and diagnosed. This study

consisted of two parts. Part A was for screening based on clinical manifestations, and Part B

was a clinical study of two commercial assays.

Figure 2. Quantitative comparison of two assays developed by commercial companies with

an in-house PCR assay. Each dot represents one urine specimen. A) In-house qPCR vs.

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QIAGEN qPCR. B) In-house qPCR vs. Shino-Test SMAP-based assay. The arrow indicates

the outlier discussed in the text.

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 Table 1. Comparison between the universal and risk-based screening approaches
this study previous study fold of enrichment
screening strategy risk-based universal

cCMV+/enrolled newborns 20/575 (3.48%)

Tokyo area 7/281 (2.49%) 16/6,256 (0.26%) 9.7
Kobe area 9/190 (4.74%) 6/1,654 (0.36%) 13.1
Nagasaki area 4/76 (5.26%) 10/3,238 (0.31%) 17.0

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 Table 2. Clinical manifestations and abnormalities among the newborns
clinical manifestations considered as number of newborns analyzed
consequece of cCMV infection CMV- (n=107) CMV+ (n=20)
fetuses
intrauterine growth restriction 20 8
ultrasound abnormality 5 6
newborns
low birth weight 38 9
premature birth 23 4
liver function abnormality 19 4
splenohepatomegaly 1 2
anemia 5 3

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jaundice that may require treatments 30 7
pneumonia/respiratory disorder 22 3
thrombocytopenia 3 6
petechiae/ecchymosis 0 5

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hearing impairment 4 6
chorioretinitis 0 1
microcephaly 7 5
hydrocephalus 2 6
meningoencephalitis 0 0
intracerebral calcification 0 6
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mother during pregnancy
fever of unknown origin 14 1
detection of CMV-specific IgM 50 (n=96) 10 (n=13)
seroconversion of CMV-specific IgG 1 (n=95) 4 (n=15)

average number of clinical manifestations*

1.7 4.1
per individual
* indications of mothers are not included
C
C
A

19

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 Table 3 Relationship of CMV-IgM-positivity with numbers of additional manifestations
numbers of cases
CMV-
cCMV numbers of manifestations other than CMV-IgM
specific IgM total
0 1 2 3
+ 10 4 1 1 4
+ - or 3 0 1 2
ND 7 2 0 5

+ 50 41 5 3 1
- - or 46 12 11 23
ND 11 1 1 9

D
+:present, -:absent, ND:not determined

TE
EP
C
C
A

20

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 Figure 1

D
TE
EP
C
C
A

21

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 Figure 2

D
TE
EP
C
C
A

22

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