We compared two protocols for extracting DNA from dried blood spots for cytomegalovirus (CMV) DNA detection and quantification by real-time PCR. Both extraction methods were reliable for the retrospective diagnosis of CMV congenital infection. Quantification of CMV DNA was valuable after normalization of viral loads with albumin gene PCR amplification results.Human cytomegalovirus (CMV) is the main organism responsible for congenital infection and permanent deafness in young children in developing countries (13). Policies for screening during pregnancy and at birth have not been implemented in European countries or in the United States, essentially because there is no well-established treatment for pregnant women or for newborns with CMV infection (7). Retrospective diagnosis of congenital infection has been achieved by PCR detection of the CMV DNA in dried blood spots (DBS) stored on perinatal Guthrie cards (2,5,6,8,9,14,16,17). Only one protocol (heat DNA extraction, followed by nested PCR) has been extensively evaluated in a clinical setting, with excellent sensitivity and specificity compared to that of viral isolation in the urine (1, 2). However, lower sensitivities (63 and 71%) were reported using the same protocol (5, 16). Alternative methods based on either phenol-chloroform or silica extraction protocols have been proposed, but their sensitivities (81% and 100%, respectively) were studied only with small numbers of patients (9, 14).In the current study, we compared two DNA extraction protocols (phenol-chloroform versus silica-based technology) followed by quantitative in-house real-time CMV DNA-specific PCR amplification (12). Indeed, knowing the CMV DNA load in DBS could provide unique insights regarding the pathogenesis and outcome of CMV congenital infection, especially the relationship between viral load and the risk of hearing loss (4, 10).DNA extraction using a whole DBS cut into thin strips with single-use scissors was performed using two protocols. In protocol one, the strips were submerged twice in 1.5 ml of washing solution (10 mM NaCl, 10 mM EDTA) for 30 min at room temperature. Then, 150 l of lysis buffer (0.32% NaOH) was added onto the strips, and the lysate was recovered after centrifugation (at 10,000 ϫ g for 2 min) and supplemented with 30 l of neutralization solution (1 M Tris, pH 7.5) before DNA extraction with a QIAamp DNA blood mini-kit (QIAGEN, Courtaboeuf, France). Protocol two was performed as described previously (15). Briefly, the strips were submerged in 400 l of extraction buffer and incubated at 56°C for 1 h. The supernatant was recovered after centrifugation and purified by phenol-chloroform extraction, followed by ethanol precipitation.CMV DNA-specific PCR amplification and human albumin PCR amplification were carried out with in-house real-time PCR assays in duplicate (11,12). The normalized value of the CMV DNA load was expressed as the number of CMV genome copies per 10 5 cells. The 45% and 95% sensitivity values of the assays were calculated with a nonlinear regression...

A new commercial real-time human cytomegalovirus (HCMV) PCR kit was evaluated after automated DNA extraction of 153 amniotic fluids in parallel with an in-house real-time PCR assay. The commercial kit displayed 100% sensitivity/specificity compared to the "in-house" assay and was suitable for prenatal diagnosis of HCMV congenital infection.The diagnosis of fetal human cytomegalovirus (HCMV) infection is usually made by amplification of HCMV genome in amniotic fluid (AF) sampled by amniocentesis. The overall sensitivity of HCMV DNA detection for prenatal diagnosis in AF by PCR ranges from 70 to 100% (13). However, the sensitivity of HCMV PCR in AF is close to 100% when using a PCR test and appropriate timing for amniocentesis (e.g., after 20 weeks of gestation and at least 6 weeks after maternal infection) (4). Nevertheless, false-negative HCMV PCR results have been reported in AF samples even under these optimal diagnostic conditions (5, 7, 13). These false-negative results were related to DNA amplification inhibition by inhibitory properties of AF (2). Moreover, although 100% specificity was reported for HCMV detection by PCR in AF (1, 3, 11, 13), rare cases of false-positive prenatal diagnosis have also been published (5, 7, 9). In these cases, although AF tested PCR positive, the AF culture was negative and the child was not infected at birth. False-positive results could be due to contamination occurring during PCR testing. This risk is higher with nested PCR, a technology that is very sensitive but exposed to contamination. Generalization of semiautomated real-time PCR methodology might help to overcome the risk of contamination and to achieve absolute specificity for HCMV prenatal diagnosis.In this study, we evaluated the performance of an automated DNA extraction system from AF samples to avoid cross-contamination between samples and to remove PCR inhibitors. We also compared the sensitivity, specificity, and quantification performance for amplification of HCMV in AF of a commercial real-time HCMV PCR assay including a PCR inhibitor detection system with our in-house real-time HCMV PCR assay (10).We tested 153 AF samples obtained from 153 women who presented with HCMV seroconversion in pregnancy and/or whose fetuses had ultrasound features compatible with HCMV infection. The samples were collected between