[Optimisation of retrospective diagnosis of cytomegalovirus congenital infection from dried blood spots].
Out of the 90% of cytomegalovirus (CMV) congenitally infected children that are asymptomatic at birth, 5 to 15% will later develop complications, mainly neurodevelopmental defects and/or deafness. Unfortunately, after the first 2 weeks of life, usual diagnostic techniques for CMV detection (viral culture and serology) are useless to differentiate congenital infection from post-natal acquired infection, whereas detection of viral DNA from dried blood spots (DBS; Guthrie cards), systematically collected from all newborns in the first days of life, has been described for late diagnosis of CMV congenital infection. The aim of our study was to choose and optimise a viral DNA extraction method from DBS and to study if CMV DNA detection is reliable when DBS are stored for 1 year at room temperature or 2 months at 37 degrees C. 10 reference cards (blood collected from CMV seronegative newborns (IgG/IgM negative) were "infected" with serial dilutions of virus and spotted on Guthrie cards) were tested. 3 extraction methods were evaluated, products of PCR were analyzes by agarose gel electrophoresis and quantification of CMV from DBS was also performed. Analysis of the results obtained from reference cards showed higher sensitivity of phenol/chloroform extraction following treatment with proteinase K, compared to heat extraction in cell culture medium or extraction with a commercial kit. We did not observe quantitative loss of viral DNA after 1 year storage at room temperature. CMV DNA detection from Guthrie cards could become a very useful tool for retrospective diagnosis of congenital CMV infection when sequelae are diagnosed in the first years of life. We are pursuing this study with DBS from congenitally infected children.
Vauloup-Fellous
C
C
Laboratoire de microbiologie immunologie biologique, hôpital Antoine-Béclère, Clamart, France. christelle.vauloup@abc.ap-hop-paris.fr
Dubreuil
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P
Grangeot-Keros
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L
D003587:Cytomegalovirus / Q000145:classification / Q000302:isolation & purification*; D003586:Cytomegalovirus Infections / Q000097:blood* / Q000175:diagnosis*; D017931:DNA Primers; D005312:Fetal Blood / Q000821:virology*; D006801:Humans; D007109:Immunity; D007231:Infant, Newborn; D016133:Polymerase Chain Reaction; D012189:Retrospective Studies; D014764:Viral Proteins / Q000235:genetics
D017931:DNA Primers; D014764:Viral Proteins