Our large, prospective study of CMV screening in newborns shows that the real-time PCR assay of both liquid-saliva and dried-saliva samples has excellent sensitivity (>97%) and specificity (99.9%) as compared with the standard saliva rapid culture. This indicates that the saliva PCR assays, which can easily be adapted for large-scale screening of newborns, will identify most infants who have congenital CMV infection.
The majority of infants with congenital CMV infection will not be identified by means of clinical examination during the newborn period. In addition, sensorineural hearing loss can develop after birth and continue to progress during early childhood in a significant proportion of children with CMV-associated sensorineural hearing loss.1,6–8,29
Thus, the availability of rapid and reliable diagnostic methods that can be adapted for high-throughput screening is essential for early identification of children at risk for CMV-associated sensorineural hearing loss. Testing dried-blood-spot specimens with the use of PCR-based methods appeared to be a promising strategy for CMV screening in newborns, because several previous studies reported that dried-blood-spot PCR assay is highly sensitive in identifying infants with congenital CMV infection.15,20,21,30
However, the results of our recent multicenter study comparing dried-blood-spot real-time PCR assays with saliva rapid culture in more than 20,000 infants revealed that dried-blood-spot PCR assays identified fewer than 40% of CMV-infected newborns.14
In addition, the performance of the dried-blood-spot PCR assay has been shown to vary according to the size of the filter-paper punch, the DNA-extraction methods, and the PCR-assay protocols used.16,22,23,31
These findings, in addition to demonstrating the challenges in developing sensitive high-throughput assays for testing dried-blood spots, suggest that many newborns with congenital CMV infection may not have detectable CMV DNA in peripheral blood. Further advances in PCR methods might improve the sensitivity of the dried-blood-spot PCR assay, however, allowing for acceptable levels of detection of infants with congenital CMV infection in the future.
The data reported here show that the same dried-blood-spot PCR protocol applied to saliva14
identified more than 97% of CMV-infected newborns. In addition, these findings show that saliva is a more reliable type of specimen than dried-blood spots for identifying congenital CMV infection by means of PCR assay and can be an effective tool for mass screening of newborns for CMV. Although testing of urine specimens collected on filter disks inserted into diapers of newborns was recently shown to be a promising approach for newborn CMV screening, urine specimen collection is not without challenges.17,32
Obtaining urine specimens from infants requires additional steps and time that are not needed for collecting saliva, and validation of methods of urine collection and urine PCR assay are needed before the practicality of urine-sample screening can be evaluated for large-scale CMV screening in newborns.
In 16 infants, saliva specimens were positive on screening by means of real-time PCR assay but not rapid culture. To determine whether these PCR results were false positives, retesting was performed with the use of PCR assay of saliva and rapid culture of saliva and urine specimens obtained at the time of enrollment into the follow-up study. If these tests were negative, we considered the screening results to be false positives. Three infants who were found to be CMV-positive only at birth, one by means of liquid-saliva PCR assay and two by means of dried-saliva PCR assay, had positive results on rapid culture and PCR assay during follow-up. These findings indicate that PCR assays identified additional CMV-infected newborns missed when tested with the use of rapid culture.
In 10 infants who had negative rapid culture results but positive PCR results (6 on liquid-saliva PCR assay and 4 on dried-saliva PCR assay), retesting yielded false positive PCR results: the follow-up saliva and urine specimens were negative for CMV. As CMV is occasionally shed in the genital tract secretions of seropositive women at delivery and in the breast milk of most seropositive mothers, these false positive results could be due to CMV-containing maternal secretions present in the infants’ saliva samples.33–38
Although false positive saliva PCR results could lead to unwarranted parental anxiety and additional testing in infants to confirm or rule out congenital CMV infection, the overall frequency of false positive results for both liquid-saliva and dried-saliva PCR assays was less than 0.03%. In addition, the small negative likelihood ratios for both saliva PCR assays indicate that a negative result on these assays does rule out congenital CMV infection ().28
Nevertheless, when saliva PCR assay is used to screen newborns, a positive screening result should be confirmed within the first 3 weeks of age to avoid false positive screening results.
The dried-saliva PCR assay failed to detect CMV infection in two newborns, leading to slightly lower sensitivity (97.4%; 95% CI, 90.8 to 99.7) than for the liquid-saliva PCR assay. Nevertheless, the simplified procedures for specimen collection, storage, and transport, combined with the high sensitivity, support dried-saliva PCR assay as a reasonable approach to CMV screening in newborns. Although the need for collection of an additional specimen adds to the complexity of the existing newborn-screening programs, the saliva PCR assays described in this study have four main advantages for CMV screening in newborns. These are reasonable sensitivity and specificity, noninvasive specimen collection, elimination of the DNA-extraction step (which simplifies the laboratory procedures, thus providing considerable cost savings), and the fact that dried-saliva specimens can be stored and transported at room temperature, further simplifying specimen handling and transport.
A limitation of this study is that the 34,812 infants found to be CMV-negative on both rapid culture and PCR assay of saliva samples obtained at the screening visit were not enrolled in follow-up to definitively exclude congenital CMV infection (by retesting with the use of rapid culture of saliva or urine). Therefore, it is possible that CMV-infected newborns may have been missed by the rapid culture, affecting our determination of the sensitivity and specificity of saliva PCR assay. However, we believe this possibility is quite low, since the saliva rapid culture has been shown to have a sensitivity of at least 98%.14,19
At present, although imperfect, rapid culture of saliva or urine specimens remains the most widely accepted standard method for identification of infants with congenital CMV infection.14,19,27
In summary, the usefulness of saliva specimens for identification of CMV by means of PCR assay was shown. The screening methods have been further simplified, with the use of dried specimens and processing that does not require a DNA-extraction step, without significant loss of sensitivity or specificity. This strategy appears to be suitable for a high-throughput assay for large-scale screening to identify newborns with congenital CMV infection.