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Study Shows Clinical Utility of qPCR for Common Viruses in Children with Acute Respiratory Illness

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With high hopes of slowing antibiotic resistance development, researchers at Karolinska University Hospital in Stockholm have demonstrated clinical utility of a qPCR assay for 16 viruses considered to be the most significant respiratory pathogens in children.

The results of the study, published in Pediatrics, showed that three of the 16 viruses are most likely to be associated with illness, but that many were also present in healthy children as well.

Distinguishing between respiratory illness caused by viruses and bacterial infections may ultimately reduce over-prescription of antibiotics. However, testing a congested, coughing child for respiratory viruses is only useful if there is a definitive link between harboring a particular virus and being ill.

To establish this link, the researchers compared the viruses in sick children under the age of five to those of healthy children sampled during the same times of year. This allowed them to conclude that certain viruses in children with acute respiratory illness are most likely to be causing disease. Other viruses were also detected in asymptomatic children, and thus may not be useful in a future qPCR-based assay or diagnostic test.

The study found at least one respiratory virus from their panel in about 72 percent of case patients and 35 percent of healthy controls. Parainfluenza virus, metapneumoniavirus, and respiratory syncytial virus had the highest relative risk for acute respiratory illness.

Lead author Samuel Rhedin told PCR Insider in an email that the group found "that the PCR detection of several of the viruses is hard to interpret due to high detection rates among asymptomatic controls." He added that there have been previous reports of respiratory virus detection in asymptomatic children using PCR, "but our study could more reliably assess the association between PCR detection and disease since we compared the cases to non-hospitalized asymptomatic controls that were matched to the cases on age and calendar time."

In addition, the study measured co-infection with multiple viruses. It found that about 20 percent of case patients harbored more than one virus from the 16-virus panel, while five percent of healthy subjects had co-infections. Respiratory syncytial virus, human adenovirus, and human bocavirus were most frequently detected in co-infection, and patients with co-infections had more severe forms of the disease.

The qPCR assay was based on one previously published in the Journal of Medical Virology in 2009. That study, also from Karolinska University Hospital, profiled 15 respiratory viruses in stored nasopharyngeal aspirates from 517 hospitalized children. The assay reportedly took four hours to perform, and ultimately fully replaced the standard immunofluorescence assay for rapid detection of viruses in the hospital's clinical laboratory.

Rhedin's group added human enterovirus to their screen, due to possible cross reactivity between probes for rhinovirus and enterovirus leading to false positive results. "For that reason we chose to run an additional PCR for enterovirus on all samples from children that turned out positive for both enterovirus and rhinovirus," said Rhedin.

The lab used Qiagen's MagAttract Virus MiniM48 kit on the Roche Light Cycler 480 platform. Rhedin said they did not evaluate other sample prep kits or platforms for this PCR method.

In the study, the authors emphasized that they did not follow subjects for consecutive sampling. Thus, they are unable to say whether viral detection in healthy controls was due to early infection, asymptomatic carriage, a low virulent infection, or prolonged shedding. The study also did not assay viral load or concurrent bacterial infection.

Although they currently have no plans to commercialize this assay, Rhedin hopes the study can lead to a clinically useful test in the future. "The most important decision physicians need to make in this patient group is whether to treat with antibiotics or not," he said. "Since bacterial diagnostics are inconclusive due to low sensitivity or specificity, they are rarely done; we believe that good viral diagnostics are important to decrease unnecessary antibiotic use."

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