Using Saliva-based PCR Assays to Detect Cytomegalovirus
New Method Comparable to Gold Standard Rapid Culture
By Genna Rollins
Cytomegalovirus (CMV) is the most common non-genetic cause of hearing loss in children, with about 10–15% of CMV-positive children eventually developing hearing deficits. Unfortunately, CMV infection typically is asymptomatic, and although a variety of methods have been evaluated to detect it, none have proven amenable to automation, precluding widespread newborn screening. Now, however, a consortium of researchers has reported developing two saliva-based polymerase chain reaction (PCR) assays for CMV, and their findings are discussed in this issue of Strategies.
Congenital CMV infection is a significant public health challenge, not only because it can cause considerable disability, but also because it is the most common non-genetic source of permanent hearing loss in the U.S. Although CMV infection is silent in most infants, a substantial portion of asymptomatic CMV-positive babies go on to experience hearing loss. Newborn hearing screening identifies some CMV-infected babies with hearing problems, but misses many others. Culture-based testing of urine and saliva samples has been the standard method to identify infants with CMV infection, but its unsuitability for automation makes this method impractical for large-scale newborn screening. The search for a robust but rapid means of detecting CMV led a consortium of researchers to explore two saliva-based PCR assays for CMV (N Engl J Med 2011;364:2111-8).
The CMV and Hearing Multicenter Screening (CHIMES) Study prospectively enrolled 34,989 infants at seven U.S. hospitals. Overall, 177 babies (0.5%) were positive for CMV according to one of three methods, rapid culture of saliva, and PCR assays of liquid-saliva and dried-saliva, respectively.
“Our study was funded by the National Institute on Deafness and Other Communication Disorders, and as part of the contract stipulation we were asked to develop diagnostic methodologies that could be applied to identify most babies with CMV infection,” recalled lead author Suresh Boppana, MD, professor of pediatrics and microbiology at the University of Alabama School of Medicine in Birmingham. “We also had to compare these newer methods with a standard method—saliva rapid culture—which is the gold standard for diagnosing babies with CMV infection.”
Last year, the consortium reported developing a PCR method using dried blood spot samples and comparing it with rapid culture, with disappointing results (JAMA 2010;303:1375-82). “Unfortunately, we found that blood spot-based PCR detects less than 40 percent of babies with congenital CMV infection, so the sensitivity was unacceptably low,” said Boppana.
In trying to identify a more sensitive method, the researchers considered both saliva and urine-based sampling, as infants with congenital CMV are known to shed virus in both fluids. “We picked saliva because it’s easier to collect, it’s non-invasive, and the collection and handling could be done relatively easily in a newborn nursery with a large number of babies,” explained Boppana.
The study protocol involved swabbing the inside of each baby’s mouth with a sterile applicator. In the case of liquid-saliva samples, the swab was placed in transport media, and shipped at 4°C to the CHIMES central laboratory at the University of Alabama. The second swab used for the dried-saliva sample was allowed to air-dry, placed in a sterile tube without transport media, and shipped to the central laboratory at ambient temperature. The samples then went directly into the PCR reaction mixture without extraction.
To improve sensitivity for the PCR assay, the researchers used two primers, one to detect the highly conserved AD-1 region of the major envelope glycoprotein B, and a second from the highly conserved 2 exon 5 region of the CMV genome. “It’s like a multiplex assay, but looking at the same, not multiple, pathogens,” explained Boppana.
The liquid-saliva assay had sensitivity and specificity of 100% and 99.9%, respectively, with positive and negative predictive values of 91.4% and 100%, respectively. The sensitivity and specificity of the dried-saliva PCR assay were slightly lower, at 97.4% and 99.9%, respectively, yielding lower positive and negative predictive values of 90.2% and 99.9%, respectively. The researchers concluded that either assay would be robust enough to use in wide scale newborn screening.
The results are very encouraging to other researchers and clinicians in the CMV field, according to Mark Schleiss, MD, professor of pediatrics, director of the division of infectious diseases and immunology, and associate chair for research in pediatrics at the University of Minnesota Medical School in Minneapolis. “The techniques and approach used in the study have helped us make great strides towards our long-term goal of identifying babies with CMV early enough after birth to implement services and interventions that will improve their outcomes,” he said. “The authors also showed that you can identify CMV from saliva with a minimum of manipulation, shortening the time to successful PCR, which is valuable information for the clinical lab. The stability they found with both liquid- and dried-saliva samples also increases the flexibility of how we might be able to store and handle the samples.”
However, Schleiss questioned how easily saliva sample collection could be implemented. “The saliva test looks to be much more sensitive, but it means collecting another body fluid sample from all newborns that currently is not collected. Nurses already are being told to do more with less and have a lot to do in discharging normal newborns. This just adds one more thing to an already complex timetable,” he cautioned.
For this reason, Schleiss expressed hope that methods of detecting CMV in dried blood spots might be improved enough for public health officials to consider those methods, especially when coupled with newborn hearing screening. “The blood spots are already procured for practically every baby born in the U.S., so it might be worth looking at them in the broader policy context,” he suggested. “The history of newborn screening is one of implementing technologies as they become available, and then as new and improved methods come along, to modify and adopt them. It’s not a static process.”
Boppana noted that other research teams are seeking to improve on the dried blood spot-based PCR method. “There’s still some hope in the scientific community that improvements can be made so it’ll be more sensitive. I think it’s possible, but I’m not very optimistic,” he cautioned. “Babies with CMV infection have a chronic inflammatory infection of variable length. We don’t know the exact onset of fetal infection, but we know from previous data that some babies with symptomatic infection don’t have detectible amounts of CMV DNA in their blood.”
Both Boppana and Schleiss hope the findings will prompt professional and other advisory groups that make recommendations to endorse newborn CMV screening. In the meantime, Boppana encouraged individual institutions to consider developing, validating, and implementing a liquid- or dried-saliva-based PCR assay for CMV.
Already, the University of Minnesota has been conducting a pilot project in collaboration with the state health department linking a dried blood spot PCR technique with newborn hearing screening results. “We’ve used it not only with babies who failed their newborn hearing screening, but also in older children who present with unexplained hearing loss. With specific parental permission and informed consent, we’ve pulled and analyzed the blood spots using a PCR technique,” he explained. “In light of the Boppana study, implementing saliva-based screening will be one of our priorities for the future.”
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