Evaluation of Different Cytomegalovirus (CMV) DNA PCR Protocols for Analysis of Dried Blood Spots from Consecutive Cases of Neonates with Congenital CMV Infections

Abstract

Two protocols for the extraction of cytomegalovirus (CMV) DNA and two methods for the amplification of CMV DNA in dried blood spots were evaluated for the retrospective diagnosis of congenital CMV infection. During the period from 1996 to 2006, a urine screening program detected 76 congenitally infected neonates. Stored Guthrie cards with blood from 55 cases and 12 controls were tested. Two spots of dried blood were cut from each card and evaluated in two centers. CMV DNA was extracted from a whole single spot. Center 1 used phenol-chloroform extraction and ethanol precipitation followed by a conventional PCR. Center 2 used the NucliSens easyMAG automated DNA/RNA extraction platform (bioMérieux) followed by a real-time PCR. For evaluation of the extraction method, DNA extracted from each blood spot was evaluated by the amplification method used by the collaborating center. The sensitivities were 66% for center 1 and 73% for center 2. None of the controls were positive. A sensitivity as high as 82% could be obtained by combining the most sensitive extraction method (the phenol-chloroform procedure) with the most sensitive PCR method (real-time PCR). The detection rate was not influenced by the duration of storage of the spots. The sensitivity was higher with blood from congenitally infected cases due to a primary maternal CMV infection, regardless of the protocol used. However, the difference reached significance only for the least-sensitive protocol (P ‫؍‬ 0.036).Cytomegalovirus (CMV) infection during pregnancy can be transmitted to the fetus, resulting in a congenital infection. Congenital CMV infection (cCMV) occurs in 0.2 to 2.2% of live-born infants. An incidence of 0.62% is found in the Brussels, Belgium, region (11).The standard diagnosis of cCMV relies on the isolation of the virus from urine or saliva collected in the first 2 weeks of life. The diagnosis of cCMV infection in children older than 2 weeks cannot be made or excluded on the basis of viral isolation. Blood is routinely collected from neonates in the first 2 weeks of life and is stored as dried blood spots (DBSs) on Guthrie cards. The detection of CMV DNA on these stored DBSs could be an opportunity to diagnose cCMV during later life, when symptoms suggestive of cCMV develop (3).The aim of this study was to test the clinical sensitivity of CMV DNA PCR with DBSs from consecutive cases of neonates with cCMV infections. We evaluated two methods for the extraction of CMV DNA and two methods for the amplification of CMV DNA from DBSs on Guthrie cards for the retrospective diagnosis of cCMV. MATERIALS AND METHODSThe study protocol was approved by the Committee of Medical Ethics of the UZ Brussel. cCMV infections. From 1996 to 2006 a neonatal screening program (11) detected cCMV in 76 newborns at the Universitair Ziekenhuis Brussels. The diagnosis of cCMV infection was made by isolation of the virus from urine within 7 days of birth. Blood from the first consecutive 57 cases and from the last 4 cases was retrieved and placed on Guthrie cards (no. 903 ...

Rapid Genotyping of Cytomegalovirus in Dried Blood Spots by Multiplex Real-Time PCR Assays Targeting the Envelope Glycoprotein gB and gH Genes

“…Detection of CMV DNA in DBS has been shown to be a challenge (3,5,6,9,10,35,36,39) due to the small amount of dried blood (50 l per spot) available. Optimizing the DNA extraction step from the DBS has been shown to result in significantly increased sensitivity of CMV DNA detection in DBS.…”

Hearing Loss and Congenital CMV Infection: A Systematic Review

“…Detection of CMV DNA can vary depending on the method of DNA extraction from the cards, the amplification method, and the part of the CMV genome being detected. [56][57][58] It may also be influenced by the time and conditions in which the cards have been stored. Cross-contamination of adjacent stored cards has been reported.…”

A pilot study using residual newborn dried blood spots to assess the potential role of cytomegalovirus and Toxoplasma gondii in the etiology of congenital hydrocephalus

“…Second, previous studies have indicated that neonatal screening for T. gondii IgM using DBS detects ~52-87% of infected newborns (Lebech et al, 1999;Paul et al, 2000;Gilbert et al, 2007). Similarly, the sensitivity of CMV DBS DNA testing approaches 100% when testing symptomatic newborns (Barbi et al, 1996;Snijdewind et al, 2012), but ranges from ~30 to 80% when testing unselected population-based newborns (Soetens et al, 2008;Boppana et al, 2010). While this suggests some case-and control-infants with T. gondii or CMV infection might have been missed with an approach that used residual DBS, the study was most likely to miss those with CMV infection who had a low viral load and thus were at low risk for clinical sequelae related to the infection.…”