Given the substantial morbidity of congenital cytomegalovirus (cCMV) infection on child health, there is considerable interest in the question of whether every newborn infant should be screened for this infection, toward the goal of identifying infants that may benefit from early intervention, anticipatory monitoring, and (in some cases) antiviral therapy [1]. Several states in the United States (U.S.) and provinces in Canada have implemented cCMV screening programs; these range from “targeted” and “enhanced targeted” screening (i.e., testing infants who have risk factors for cCMV, such as “referral” status on their newborn hearing screen (NHS) or other signs/symptoms of infection) to universal testing of all newborns [2]. A challenge in implementation of universal screening is the issue of what clinical specimens are ideal for testing the newborn infant. Obtaining a urine sample on every infant prior to hospital nursery discharge holds the pride of place as the “gold standard” for detection of cCMV, but collection of urine samples on all newborns is labor-intensive and cost-prohibitive. Implementation of cCMV screening based on amplification of CMV DNA in dried blood spots (DBSs) collected as a standard of newborn screening (performed to test for metabolic and genetic diseases) has emerged as a cost-effective option that can be readily implemented into standard newborn care [3,4], but questions have been raised about whether the sensitivity of this technique is sufficiently robust to make this the preferred approach to newborn cCMV screening [5]. An alternative and easy-to-obtain specimen is saliva, and many studies have examined the utilization of saliva specimens for cCMV newborn screening [6]. Although this technique is highly sensitive [7], it has been criticized as a problematic methodology because of the high numbers of false-positive tests that are reported in screening programs.
Against this backdrop, an interesting and valuable paper by Santander and colleagues in the current issue of the International Journal of Neonatal Screening addresses the issue of saliva-based universal newborn cCMV screening [8]. This group leveraged the recent implementation of, and data from, a universal DBS screening in Ontario, Canada [4], to compare the relative diagnostic accuracy of DBS to saliva—specifically, dried saliva specimens spotted on Whatman 903 filter paper—for newborn cCMV screening [8]. The study design was a prospective paired-sample pilot that studied consecutive births at 10 birth centers over a ~15-month period. Newborns that tested positive on either specimen were then then evaluated by urine CMV PCR to either confirm—or refute—the diagnosis of cCMV.
What did this study find? Among 45,597 births, approximately 40,000 infants had interpretable results for both DBS and dried saliva specimens. Of 150 newborns who screened positive, 39 underwent confirmatory urine testing in the first 21 days of life, and 24 had proven cCMV infection. As a percentage of DBS sensitivity, dried saliva specimen screening demonstrated not only a non-significant relative sensitivity (rSe) of 109% [95 CI: 97–123%] but also a relative false positive rate (rFPR) that was 700% [95 CI: 175–2795%] of the DBS rate. The observed 700% rFPR with the DSS-based strategy aligns with Minnesota’s recent comparison of a dried saliva swab-based cCMV test to a DBS-based diagnostic approach, which found saliva generated false positive results at an 8–10-fold greater rate than did the DBS assay [3]. Thus, in this pilot study of dried-saliva-based screening for cCMV, the authors concluded that not only did saliva screening fail to significantly increase sensitivity for cCMV, but it also had an increased false positive rate [8]. The authors noted that the performance profile of saliva-based screening did not support population-wide implementation of a saliva-based approach for universal cCMV screening.
The lack of enhancement of sensitivity of saliva-based screening compared to DBS screening in the Santander study was surprising [8]. However, in the primary analysis of screening performance, where infants confirmed within 21 days of life were evaluated, there was no gain in sensitivity using dried saliva. Most experts have opined that DBS-based screening is inferior to saliva-based screening. Some experts have expressed skepticism about the sensitivity of DBS screening [5], but it is important to note that in the only other recent study directly comparing saliva with DBS PCR, although the analytic sensitivity of DBS using two primer pairs for identification of CMV target sequences only approached ~80% [3], the clinical sensitivity of DBS PCR—that is to say, the sensitivity of the DBS assay in identifying children with actionable cCMV disease or sequelae—approached 90% [9]. This data, as well as the data from the Santander and colleagues [8], strongly support implementation of policies that would codify universal cCMV screening using DBS testing. Such an approach should be substantially less expensive for states, provinces and municipalities, since the assays would leverage a sample that is already collected as a standard of newborn care and would utilize facilities and infrastructure already in place for newborn screening programs [2,6]. With no enhancement of diagnostic sensitivity conferred by dried saliva testing as noted in the Santander study, there is reason to believe that there may be no clear advantage to the use of the dried saliva approach for universal cCMV screening compared to the DBS.
The most likely presumed source of CMV DNA in the dried saliva samples from the Santander study is from breast milk (or, more likely, colostrum). CMV is ubiquitous in colostrum, transitional milk, and breast milk in seropositive lactating mothers [10], and indeed it is even present in commercial donor milk [11]. Although donor milk is not a source of acquisition of CMV infection, its use can render a dried saliva test falsely CMV PCR-positive for an infant receiving donor milk feeds in the newborn nursery. Proponents of saliva-based cCMV screening note that the likelihood of a false-positive dried saliva PCR may be reduced by maximizing the time interval between nursing or feeding the infant and obtaining the saliva swab; commonly, it is recommended that this interval should be at least 30–60 min. However, there is no compelling evidence that this approach substantially reduces the number of false positive tests. Moreover, although the Santander study focused on the clinical utility of saliva testing for universal cCMV screening, it is important to recognize the harm that can be done by using saliva-based PCR in the evaluation of infants that test falsely positive for CMV DNA through saliva-based hearing-targeted or expanded-targeted screening [2] approaches. A positive saliva test should engender a reflex order for urine CMV PCR testing to confirm or refute the PCR test, which should be performed before hospital discharge, but logistically there are many barriers. Newborn infants who refer on the initial newborn hearing screening are typically tested a second time before hospital discharge and, if they refer a second time on the screening test, they need follow-up audiological evaluation. At that point in a term newborn’s hospital stay, there simply often is not enough time to obtain both a saliva CMV PCR and a follow-up urine sample, since families and staff alike are poised to discharge the infant home. A false-positive saliva PCR test—without a confirmatory urine sample—can cause harm by generating undue anxiety on the part of both clinician and parent alike, and lead to unnecessary diagnostic testing of the newborn.
Another important complicating variable is that the discharge instructions that the family of a newborn receives after a positive saliva screen often involve returning to an outpatient care facility for a follow-up, definitive urine sample for CMV PCR—since there are logistical barriers to completing the urine sample before discharge. This outpatient urine collection must be done prior to 21 days of age, but this is often a burden that many families simply cannot meet. The algorithm immediately disenfranchises families that may not have the means to obtain follow-up outpatient care in this time window, and in the process, it creates a huge health equity challenge. This approach is also very labor-intensive, requiring the acquisition and processing of multiple specimens and arrangement of complex follow-up instructions. The costliest aspect of health care (at least in the U.S.) is the cost of a provider’s time, and this testing algorithm is time-consuming and, hence, costly. As noted above, incorporating the cost of CMV DBS PCR into the existing infrastructure of a state health department’s newborn DBS screening program represents a much less expensive alternative for cCMV screening compared to saliva screening, with a minimum loss of diagnostic sensitivity.
In summary, the paper from Santander and colleagues in Ontario provides strong support for expansion of DBS-based universal cCMV screening. Continued efforts need to be made to optimize the sensitivity of DBS testing, but the logistical, economic, and practical barriers provided by saliva-based screening programs are considerable and, given the false-positive rate with questionable enhancement of sensitivity, may actually cause more harm than good. The thoughtful design of the Santander study also demonstrates that it is imperative that future “roll-out” of universal screening programs, particularly of DBS testing, include proper control groups and careful assessment of comparators for newborn CMV testing to inform and direct optimized screening in the future. Recent papers that have examined the utility of saliva-based universal screening for cCMV have shown progress in this arena [12,13,14], and show promise for the future, but it is not yet clear that a single low-level positive identification of CMV DNA detection in a saliva sample (below a pre-defined “cut-off” level), can be written off as a “false positive”. Until more knowledge is available, low-level positives should probably still have a “gold standard” urine CMV PCR detection assay performed. A pooled saliva screening strategy reported by Merav et al. demonstrates excellent sensitivity and specificity, and merits consideration for widespread implementation [15]. However, given the concerns about false-positive saliva PCR, the attendant costs of saliva testing to the health care system, the complexities of test interpretation, and the significant health equity concerns, state health departments should probably focus on wider implementation of DBS-based universal cCMV screening in the U.S. Universal DBS screening avoids false-positive tests, demonstrates an acceptable clinical sensitivity, and importantly ensures more equitable follow-up for infants with cCMV infection.
Funding
The author acknowledges support from the National Institutes of Health, Eunice Kennedy Shriver National Institute of Child Health and Human Development, R01 HD118989.
Data Availability Statement
No new data were created or analyzed in this study. Data sharing is not applicable to this article.
Conflicts of Interest
The author declares no conflicts of interest.
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