By Ben Butkus
A group led by clinical researchers from the University of Alabama-Birmingham has developed a real-time PCR assay that can detect cytomegalovirus in both liquid and dried saliva specimens from newborns with near-100 percent sensitivity and specificity when compared to the gold standard of rapid culture from saliva, according to research published today.
In doing so, the researchers — who previously demonstrated that PCR-based CMV testing of commonly collected dried blood spots had low sensitivity compared to rapid culture — have made a strong case for saliva-based testing as a more promising tool to screen newborns for CMV, a leading cause of hearing loss in children.
However, the test now needs to receive further validation and a recommendation by one or more professional medical organizations in order to be implemented in newborn CMV screening programs nationwide, the study's lead author said this week.
In the multicenter study, funded by the National Institute on Deafness and Other Communication Disorders and published today in the New England Journal of Medicine, UAB researchers Suresh Boppana, Karen Fowler, and colleagues conducted rapid culture and real-time PCR testing on both liquid and dried saliva samples obtained from 34,989 newborns at seven US hospitals.
The researchers obtained saliva samples from the newborns by gently rubbing a synthetic polyester fiber tip swab inside their cheeks in order to saturate it with saliva. For liquid saliva PCR testing, the sample was placed in transport medium, shipped to the testing facility using cold storage, vortexed, and placed directly into a PCR reaction chamber. For dried saliva testing, the swab was allowed to dry, placed in a tube without media, shipped to the testing facility at room temperature, rehydrated, and then tested in the same manner as the liquid saliva sample.
Of 17,662 newborns screened with the liquid saliva PCR assay, 17,569 were negative for CMV, and the remaining 85 were positive using both rapid culture and PCR. Meantime, of 17,327 newborns screened using the dried saliva PCR assay, 74 were positive for CMV using PCR compared to 76 positives using rapid culture. The overall sensitivity and specificity of the liquid-saliva test were 100 percent and 99.9 percent, respectively; while the sensitivity and specificity of the dried-saliva test were 97.4 percent and 99.9 percent respectively.
"The results are comparable, although the sensitivity of the liquid PCR was 100 percent; whereas the dried saliva PCR was about 97 percent, because we missed a couple of babies. I'm not exactly sure why," Boppana told PCR Insider this week. "But at the same time we picked up a baby that was not detected in rapid culture, which was our gold standard assay."
Boppana added that the dried saliva test would be preferable for large-scale screening "for the ease of storage and transport; however the liquid saliva test would work, as well."
Although tissue culture-based fluorescent detection of virus antigen from saliva or urine specimens is the gold standard for newborn CMV screening, it is not amenable to mass screening because it is labor- and resource-intensive and requires tissue culture facilities.
"Most babies infected with CMV don't show symptoms at birth," James Battey, director of NIDCD said in a statement. "It's important for us to develop diagnostic tools to screen babies for congenital CMV infection so that those who test positive can be monitored for possible hearing loss and, if it occurs, provided with an appropriate intervention as soon as possible."
In the NEJM study, the researchers used a two-color real-time PCR assay, developed at UAB, that included primers to detect the highly conserved AD-1 region of the major envelope glycoprotein B; as well as a second primer set from the highly conserved immediate-early 2 exon 5 region. The assay used Absolute qPCR Low ROX Mix from Thermo Scientific's AbGene business and a Life Technologies Applied Biosystems 7500 real-time PCR system.
The assay protocol was nearly identical to that used in a previous study published by the group last year in the Journal of the American Medical Association, which PCR-tested dried blood spots collected from more than 20,000 infants and compared the results to rapid culture. The only major differences between the two protocols were that the DBS study required DNA extraction whereas the saliva study did not; and for part of the DBS study the researchers used only a single-primer assay.
The surprising conclusion of the previous study, which Boppana and colleagues claim was the first large-scale, prospective study comparing DBS PCR testing to gold standard, was that the single-primer assay had a sensitivity of about 28 percent while the dual-primer assay yielded sensitivity of 34 percent — both far too low to be considered in mass screening campaigns (PCR Insider, 4/15/10).
This result was somewhat unfortunate seeing as how dried blood spots are already commonly collected from newborns to test for other diseases and disorders. However, the high sensitivity and specificity of the saliva-based tests — not to mention their simplicity and time savings due to the elimination of the DNA-extraction step — suggest that they could be used in lieu of DBS testing in a large-scale newborn screening campaign, according to the researchers.
"If the blood spots had worked, that would have been great, because it's easier to incorporate," Boppana said. "At the same time, I think in the last 15 to 20 years … a hearing screening program … has been added to newborn screening across the country. So that's a precedent — if it is a significant issue, it could be done. At least our study shows that we have an assay that could be adapted to an automated and [high-throughput] fashion."
Boppana said that he believes the reason for the discrepancies in sensitivity between DBS and saliva testing have to do with extremely low levels of CMV in the blood, but high levels of the virus in the saliva of infected newborns. "It has been known for some time that saliva contains large amounts of the virus" in infected newborns, he said.
However, he noted that others in the field believe that the DBS PCR assay could be improved by using different methods to improve sensitivity, such as collecting a larger DBS or improving DNA extraction efficiency. "It's possible that you could have a DBS PCR test be sensitive enough, but I'm not optimistic," Boppana said.
Nevertheless, implementing a saliva-based test in a large-scale newborn screening campaign will be difficult because it would require that physicians perform saliva collection in addition to DBS collection, which isn't likely going away anytime soon.
In order to scale this obstacle, Boppana said, the test would likely need to receive a recommendation from "some sort of agency at the national level." Such professional organizations might include the American College of Medical Genetics and the American Congress of Obstetricians and Gynecologists; or even government agencies such as the National Institutes of Health or the US Centers for Disease Control and Prevention, Boppana said.
The UAB Research Foundation is currently "looking into" patent protection for the saliva-based PCR test for CMV, and is also seeking potential commercial partners for the assay, Boppana added. The researchers are now investigating how much congenital CMV infection contributes to overall hearing loss at birth and between the ages of 3.5 to 4 years by enrolling infants who tested positive in a follow-up program to monitor their hearing.
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