Evaluation of Cytomegalovirus (CMV) DNA Quantification in Dried Blood Spots: Retrospective Study of CMV Congenital Infection

Abstract

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...

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.…”

The “Silent” Global Burden of Congenital Cytomegalovirus

“…DBS also have appeal for use in newborn congenital CMV screening programs. However, the variable and disappointing rates of detection of CMV DNA (34 to 100%) have made DBS unsuitable for these purposes (112,114,(116)(117)(118)(119)(120)(121)(122), possibly because not all newborns are viremic at birth or due to technical factors (118,(122)(123)(124). However, a positive DBS PCR finding is diagnostic of congenital CMV infection and accordingly can be useful to retrospectively diagnose congenital CMV infection beyond the neonatal period.…”

“…Dried blood spots (DBS), or Guthrie cards, which are collected routinely at birth in certain countries for newborn genetic and metabolic diseases screening, can be stored for extended periods of time. Since CMV DNA is stable on such DBS cards for up to 18 years, they offer an attractive tool for the retrospective molecular diagnosis of congenital infection in individual children who present with delayed-onset sequelae (112)(113)(114)(115). DBS also have appeal for use in newborn congenital CMV screening programs.…”

Quantitation of Cytomegalovirus DNA Load in Dried Blood Spots Correlates Well with Plasma Viral Load

“…Several previous studies described the use of dried blood spots (DBS) for detection of CMV (2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12). However, most of these prior studies using DBS for detection of CMV focused on children with congenital CMV infection, where quantitation and monitoring of viral load over time are not routinely performed.…”