Diagnosis of congenital cytomegalovirus infection by detection of viral DNA in dried blood spots

Abstract

Background: The reference method of cytomegalovirus (CMV) isolation from urine or saliva is not a feasible routine technique for all newborns, and laboratory diagnosis of this infection would be useful both for epidemiological purposes and to enable prompt institution of adequate measures to identify and correct late sequelae. Extraction and amplification of viral DNA from dried blood spots (DBS) collected from babies in the first days of life during routine screening for genetic and metabolic disorders has been proposed for the early diagnosis of viral congenital infections. Objectives: To test the method for CMV DNA extraction from DBS and to evaluate the results obtained in newborns with and without a diagnosis of congenital infection based on viral isolation from urine and or saliva at birth. Study design: DBS from Guthrie cards collected in babies who underwent virological tests for CMV infection were tested for CMV DNA by observers blinded to the virological results. DNA was extracted from DBS both in water and in cell culture medium according to Shibata et al. with minor modifications. The products of nested polymerase chain reactions (PCR) amplifying two regions in the IE1 and gp58 genes were detected by agarose gel electrophoresis. Strict control measures were adopted to avoid carryovers and contaminations. Results: DBS from the eight symptomatic and 11 asymptomatic congenitally infected babies were positive when extraction was performed in medium, whereas extraction in water failed to identify two of the asymptomatic cases. The results obtained with the two extraction methods agreed in the remaining cases; the 71 CMV negative control babies were negative and two out of 21 cases of supposed postnatal infection were diagnosed as congenital on the basis of a positive DBS. All positive cases were identified by gp58 PCR but only slightly over half of them by IE1 PCR. Extraction in medium was more efficient than in water. Conclusions: The method of CMV DNA extraction in medium followed by amplification of the gp58 region showed 100% sensitivity and specificity compared with isolation in cell culture. Therefore, we propose this procedure to diagnose congenital CMV infection at birth and also later.