IMPROVED PRENATAL-DIAGNOSIS OF CONGENITAL HUMAN CYTOMEGALOVIRUS-INFECTION BY A MODIFIED NESTED POLYMERASE-CHAIN-REACTION

Two major variables may cause false-negative results in prenatal diagn osis of congenital human cytomegalovirus (HCMV) infection: sensitivity of the technique(s) used; and time elapsed between maternal infection and antenatal testing. Previous results indicated that rapid HCMV iso lation from amniotic fluid samples and viral DNA detection in amniotic fluid by nested polymerase chain reaction (nPCR) had comparable level s of sensitivity (69.2% and 76.9%, respectively). The nPCR protocol wa s reviewed follow ing two additional false-negative antenatal diagnosi s in a twin pregnancy during which two procedures were performed at 18 and 23 weeks of gestation, respectively. in the new assay, multiple ( instead of single) and 100 (instead of 20) mu l amniotic fluid aliquot s were individually amplified and tested by nPCR. By using this approa ch, low DNA levels (1-10 genome equivalents) were detected in 1-5/8 re plicates of amniotic fluid samples taken from both twins during both p rocedures. In addition, viral DNA was detected in 5/6 replicates from two amniotic fluid samples still available from two previous false-neg ative cases. However, nPCR on multiple amniotic fluid replicates did n ot anticipate positive prenatal results in a retrospective case, which required two procedures for correct diagnosis and, when prospectively employed, did not avoid one additional false-negative prenatal diagno sis 8 weeks after maternal infection. Thus, delayed intrauterine trans mission of the infection may be a potential cause of false-negative re sults. However, the combination of a very sensitive technique with app ropriate timing of prenatal testing can substantially increase the rel iability of prenatal diagnosis results. J. Med. Virol. 56:99-103, 1998 . (C) 1998 Wiley-Liss, Inc.