Characterization of a universal screening approach for congenital CMV infection based on a highly-sensitive, quantitative, multiplex real-time PCR assay.

Angela Nagel,Dariusz Michna,Klaus Überla,Klaus Korn,Thomas Lücke,Emmanouela Dimitrakopoulou,Khalid Shahada,Norbert Teig,Peter Kern,Katrin Neumann,Philipp Steininger

doi:10.1371/journal.pone.0227143

Abstract

The majority of congenital cytomegalovirus (cCMV) infections are asymptomatic at birth and therefore not diagnosed. Approximately 10–15% of these infants develop late-onset hearing loss and other developmental disorders. Implementation of a universal screening approach at birth may allow early initiation of symptomatic interventions due to a closer follow-up of infants at risk and offers the opportunity to consider treatment of late-onset disease. Real-time PCR assays for the detection of CMV DNA in buccal swab samples demonstrated feasibility and good clinical sensitivity in comparison to a rapid culture screening assay. Because most cCMV infections remain asymptomatic, a universal screening assay that stratifies CMV infected infants according to low and high risk of late-onset cCMV disease could limit the parental anxiety and reduce follow-up costs. We therefore developed and characterized a screening algorithm based on a highly-sensitive quantitative real-time PCR assay that is compatible with centralized testing of samples from universal screening and allows to determine CMV DNA load of saliva samples either as International Units (IU)/ml saliva or IU/105 cell equivalents. 18 of 34 saliva samples of newborns that tested positively by the screening algorithm were confirmed by detection of CMV DNA in blood and/or urine samples obtained during the first weeks of life. All screening samples that could not be confirmed had viral loads of <2.3x105 IU/ml saliva (median: 6.8x103) or 1.3x105 IU/105 cell equivalents (median: 4.0x102). The viral load of screening samples with confirmed cCMV infection ranged from 7.5x102 to 8.2x109 IU/ml saliva (median: 9.3x107) or 1.5x102 to 5.6x1010 IU/105 cell equivalents (median: 3.5x106). Clinical follow-up of these newborns with confirmed cCMV infection should reveal whether the risk of late-onset cCMV disease correlates with CMV DNA load in early life saliva samples and whether a cut-off can be defined identifying cCMV infected infants with or without risk for late-onset cCMV disease.

Highlights

Identification of connatal cytomegalovirus infections is key to prevent or mitigate the sequelae of symptomatic or asymptomatic cCMV infections by antiviral therapy, intensified monitoring and/or early initiation of supportive therapy
In a study exploring the influence of pre-analytic factors, this approach has revealed the best CMV DNA recovery efficiency compared to a viral transport medium with or without prior DNA extraction ([13], S2 Table)
Detection of Herpesvirus saimiri (HVS) in appropriate amounts was assessed in all CMV DNA negative samples whose albumin DNA levels exceeded 50 copies per Polymerase chain reaction (PCR) reaction to exclude false negative findings resulting from PCR inhibition

Summary

Introduction

Identification of connatal cytomegalovirus (cCMV) infections is key to prevent or mitigate the sequelae of symptomatic or asymptomatic cCMV infections by antiviral therapy, intensified monitoring and/or early initiation of supportive therapy. The targeted screening of congenital CMV infection provides screening for newborns with a history of maternal primary infection during pregnancy, a failed hearing screening, or other symptoms suggestive of cCMV infection [1,2,3,4] This strategy would not identify a substantial percentage of newborns with asymptomatic cCMV infection approximately 10% of which are at risk for lateonset cCMV disease. While these cases could be identified by screening of all newborns for cCMV infection, 90% of the asymptomatic cCMV infections identified by such a universal screening would not result in late-onset cCMV disease, but would lead to years of intensified monitoring and severe parental anxiety of the affected children. We evaluated a real-time PCR assay controlling PCR inhibition and simultaneously quantifying CMV DNA and human genomic DNA from buccal swab samples of neonates

Material and methods

Evaluation of results and statistical analysis

Results

Discussion