By Ben Butkus
This story was originally published on April 13.
Real-time PCR-based testing of dried blood spots is not a viable option to screen newborns for a virus that causes hearing loss because it does not have sufficient sensitivity, according to the results of a study published today in the Journal of the American Medical Association.
Despite this, PCR testing of other bodily fluids such as saliva still holds promise as a routine test for the infection in newborns, according to the study's lead author and other experts.
The study focused on cytomegalovirus, an important cause of congenital infection and a leading cause of sensorineural hearing loss in children. Of the estimated 20,000 to 40,000 infants born each year with congenital CMV infection in the US, 90 to 95 percent have no detectable clinical abnormalities at birth and thus are not identified by routine clinical examination.
To identify at-risk infants early in life, "rapid, reliable, and relatively inexpensive methods to screen newborns for congenital CMV are needed," the researchers wrote in their paper. "Identification of children at increased risk of CMV-associated SNHL early in life will allow targeted monitoring … in order to intervene at critical stages of acquiring speech and language skills."
Currently, tissue culture-based fluorescent detection of virus antigen from saliva or urine specimens is the most popular method for newborn CMV screening. However, this technique is not amenable to mass screening because it is labor- and resource-intensive and requires tissue culture facilities.
PCR, on the other hand, is much better suited for high-volume and –throughput screening, and is generally less expensive. PCR can be used to test a variety of newborn specimens, including saliva, urine, and DBS; however, since DBS are collected routinely for metabolic screening of all infants in the US, there has been considerable push to develop a PCR test using those specimens.
The new study, led by Suresh Boppana, a doctor in the University of Alabama-Birmingham department of pediatrics, sought to provide the first direct comparison of DBS real-time PCR assays with fluorescent detection of virus antigen from saliva or urine specimens, which is considered the gold standard for newborn CMV screening.
Boppana told PCR Insider this week that a number of papers have been published claiming a high degree of sensitivity of DBS PCR assays. However, most of these papers have examined a selected infant population, and none has compared the results of DBS PCR assays with a standard method, such as tissue culture.
Most studies, Boppana said, have examined infants that had already been screened as positive for CMV infection and then tested them with the PCR method.
"They don't have a denominator," he said. "They don't know how many [positives] are being missed. I don't know how you can come up with an accurate sensitivity of an assay if you don't compare it to the gold standard. I believe we are the first to compare PCR with a standard screening."
As described in the JAMA study, between March 2007 and May 2008, Boppana and colleagues tested samples collected from infants born at seven US medical centers. Upon enrollment, saliva specimens were collected from participating infants along with blood spots obtained at the time of metabolic screening.
For each DBS, the researchers extracted DNA using a Qiagen M48 robotic system with MagAttract technology; and detected CMV DNA using a Life Technologies Applied Biosystems 7500 real-time PCR system and Absolute qPCR Low ROX Mix from Thermo Scientific's AbGene business.
During the first 10 months of the study, the real-time PCR assay included primers to detect the highly conserved AD-1 region of the major envelope glycoprotein B. During the final five months of the study, the method was modified to include a second primer set from the highly conserved immediate early 2 exon 5 region, in an effort to improve its sensitivity. Boppana and colleagues at UAB developed both of the assays.
Congenital CMV infection was confirmed in 92 of 20,448 infants, and 91 of those had positive results using saliva rapid culture method. Of the 11,422 infants screened using the single-primer DBS PCR, 17 of 60, or 28 percent, had positive results. Meantime, of the 9,026 infants screened using the dual primer assay, 11 of 32, or 34 percent, screened positive.
The researchers surmised from their data that as many as 80 percent of infants with congenital CMV infections could be missed using PCR, even when the two-primer method is employed.
"Basically it's not very detectable based on PCR methodologies that are available right now," Boppana said. "It's kind of disappointing because it's a negative study, but it's not surprising."
However, the problem might not be with the PCR methods. Indeed, Boppana and colleagues vigorously tested and compared their DNA extraction and amplification procedures with other methods. "I don't think this is what is causing our results," he said.
Instead, Boppana said that the use of DBS is likely the difference maker. "It's probably not the way to go," he said.
The fact that DBS is already a mandatory procedure, coupled with the results of several published studies showing that PCR of peripheral blood can be used to detect invasive CMV infections in immunocompromised adults, caused researchers to push for a PCR-based test using infant DBS samples.
However, as the researchers wrote in the JAMA paper, the pathogenesis of congenital CMV infection is likely to be different from that in immunocompromised hosts. Indeed, these patients usually experience acute CMV infection of symptomatic reactivation before blood-based PCR testing.
Meantime, congenitally infected infants may have acquired CMV infection months before birth and are no longer viremic when tested. "We really have no way of knowing how long they've been infected," Boppana said.
However, there is still hope for developing a rapid, inexpensive PCR-based screen for congenital CMV infection. "There are other possibilities for PCR testing, like saliva or urine samples," Boppana said.
In fact, his research group is currently examining PCR-based testing for CMV from infant saliva samples. Although their work is not finished, and they are just starting to prepare a manuscript for publication, Boppana said that the early results "look quite promising."
Supporting this idea, in an accompanying editorial published today in JAMA, James Bale of the University of Utah School of Medicine wrote that the findings of Boppana and colleagues do not necessarily suggest that the goal of universal screening for CMV should be abandoned.
"Although current tissue culture methods are too time consuming and expensive to be used for mass screening of newborns, molecular detection methods can provide high-throughput screening for CMV," Bale wrote.
"While assaying the newborn dried blood spot may not be the solution … detecting viral DNA in specimens obtained in the first few days of life may still be possible," he added. "Rather than assaying blood, one possible approach is to consider assaying a specimen of the infant's saliva. The saliva of virtually all congenitally infected infants contains abundant quantities of CMV DNA that can be detected by current PCR methods."