The problem

Congenital cytomegalovirus (cCMV) infection is the most common intrauterine infection worldwide; it can lead to neurodevelopmental disabilities and hearing loss in the infant, which can manifest at birth or develop later during childhood1. At present, targeted screening of cCMV infection by PCR testing of saliva samples is used in many centers, focusing on infants with failed newborn hearing screen, clinically suspected cCMV infection, or a history of maternal CMV infection2,3. However, targeted screening of high-risk neonates misses most infants with cCMV who are asymptomatic at birth, yet are at risk of late-onset sequelae3,4. Thus, universal newborn screening of cCMV has been increasingly advocated to facilitate timely diagnosis and management2,3. In the absence of a high-throughput screening test that can identify all infected neonates, the development of an accurate and efficient testing strategy for universal cCMV screening has remained an ongoing challenge.

The solution

A promising approach is sample pooling, in which a set number of samples is pooled and tested together: if the pooled sample is negative, all the individual samples within are considered negative, and if the pooled sample is positive, all its samples are re-tested individually (Fig. 1a). The expected efficiency of pooling (the total number of samples tested per single PCR test) depends on the prevalence rate of the measured parameter: the lower the prevalence, the higher the efficiency. We therefore hypothesized that the low birth prevalence of cCMV (average 6.4 per 1,000)1 would make it an ideal candidate for pooled screening. In addition, because cCMV is characterized by a high viral load in saliva, the loss of sensitivity after sample dilution by pooling should not be a major theoretical concern. On the basis of the saliva viral loads among individuals with cCMV whom we had previously individually tested, we predicted a detection sensitivity of 99.5% for an 8-sample pooling. Here, building on our pooling pipeline that we successfully implemented during the COVID-19 pandemic5, we developed a new pooling setup for cCMV detection and assessed it for universal screening of cCMV in two hospitals from April 2022 to April 2023.

Fig. 1: Newborn cCMV screening scheme and performance rates.
figure 1

a, Overall scheme of the newly implemented pooled testing, in which up to eight saliva samples are pooled for a single PCR test; when the pooled sample is positive, individual saliva and urine PCR tests are performed as indicated. b, The newborn screening rates during targeted testing (January 2017–December 2021), our initial pilot of pooled testing (January–March 2022), and since the implementation of universal pooled screening (April 2022), eventually reaching approximately 94% of live neonates. © 2024, Merav, L. et al., CC BY 4.0.

Full size image

During this period, 15,805 infants (93.6% of all live neonates) were screened for cCMV using the pooled approach that has since become our routine screening method (Fig. 1). The empirical efficiency (number of tested neonates per single PCR test) of the pooling was 6, which means that 83% of the saliva tests that would have been required if testing each sample individually were no longer necessary. PCR loss of sensitivity was minor, in accordance with the theoretical prediction for an 8-sample pool. cCMV was identified in 54 neonates, with a birth prevalence of 3.4 per 1,000. More than half (55.6%) of the infants with cCMV were asymptomatic at birth and would have been missed by targeted screening.

The implications

Our findings demonstrate the feasibility and benefits of pooled-saliva testing as an efficient, cost-sparing and sensitive approach for universal screening of cCMV and support its widespread implementation. The pooling setup can be easily integrated in any medium-to-large medical laboratory, and parental acceptance of cCMV screening was high, mitigating early concerns. Our finding that in the absence of universal screening most cCMV cases would have remained undiagnosed underscores the clinical implication of universal screening toward early diagnosis, monitoring and potential treatment of cCMV, and it further highlights the importance of universal screening in defining the accurate population prevalence of cCMV.

Because the study population is limited to two hospitals, our estimate of cCMV prevalence might not be representative of the entire country population. Of note, the empirical efficiency of pooling might vary between populations with different rates of cCMV prevalence. In addition, although we showed that single cases (0.5% of positive cases) might be missed by 8-sample pooling, large-scale implementation of the pooled assay at multiple centers and in larger populations might miss larger numbers of cases.

Data derived from the implemented universal screening will serve to define the burden, risk factors and long-term clinical outcomes of cCMV. Because cCMV prevalence has been shown to vary considerably by race, ethnicity and maternal seroprevalence1,3, it will be important to further assess the prevalence of cCMV in distinct subpopulations.

Dana G. Wolf1 & Moran Yassour2

1Hadassah Hebrew University Medical Center, Jerusalem, Israel. 2The Hebrew University of Jerusalem, Jerusalem, Israel.

Expert opinion

“Cytomegalovirus is the most common intrauterine infection, yet it is only found in single-digit cases per thousand neonates tested. Universal screening is desirable for timely diagnosis and potential antiviral therapy. However, testing individual saliva samples produces a vast number of negative results and a few false-positive ones. Here, the authors describe a way of minimizing screening costs while reducing the number of false-positive results and demonstrate the efficiency of a pooling approach for universal screening.” Paul Griffiths, University College London, London, UK.

Behind the paper

This study was motivated by three main factors. First and foremost, we were driven by the unmet clinical need to identify all neonates with cCMV, so that early treatment and monitoring could be delivered to a large proportion of infants who are currently missed. Second, this project was facilitated by the newly available pooled diagnostic approach (which we have implemented to cope with the vast number of tests during the COVID-19 pandemic), which made universal screening of cCMV feasible. Finally, an essential component of this study was the close collaboration of experts in virology, data processing, neonatology and obstetrics. Together, the resulting screening rate of cCMV, approaching 94% of all neonates at our centers, along with the high real-world efficiency and sensitivity of the pooled-saliva tests, represent the most important achievements of the research. D.G.W. & M.Y.

From the editor

“Although cCMV infection remains one of the leading causes of congenital disease, the development of an accurate and efficient testing strategy to identify all affected infants has remained difficult. This paper presents a cost-effective, accurate and non-invasive method for cCMV testing, which could revolutionize the implementation of universal screening.” Editorial Team, Nature Medicine.