Evaluation of different cytomegalovirus (CMV) DNA PCR protocols for analysis of dried blood spots from consecutive cases of neonates with congenital CMV infections.

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).