Prenatal diagnosis of congenital toxoplasmosis by duplex real-time PCR using fluorescence resonance energy transfer hybridization probes

The diagnosis of congenital toxoplasmosis frequently relies on PCR tests of amniotic fluid (AF). A duplex real-time quantitative PCR test based on flu orescence resonance energy transfer was developed to quantify the parasite load and to decrease the risk of contamination. An internal control based o n the detection of 10 pg mouse DNA added to the AF was included to check fo r PCR efficiency. The relationship between the parasite load and the occurr ence of ultrasonographic abnormalities in 87 samples of AF was analyzed. Se ven AF (8%) had a parasitic load > 10(3); 14 (16%) had > 10(2)-less than or equal to 10(3); 26 (30%) had > 10-less than or equal to 10(2); and 40 (46% ;,) had less than or equal to 10 parasites/ml. Four of the six AF with cere bral ventriculomegaly had > 10(3) parasites/ml. The other two had 130 and 2 4 parasites/ml, respectively. No parasitic loads of >10(3) parasites/ml and no ultrasonographic abnormalities were observed in the 11 AF with maternal toxoplasmosis in the third trimester. Therefore, there is a trend to assoc iate high parasite count with ultrasonographic abnormality, but the main co ncern remains early maternal infection. The importance of quantification sh ould be better evaluated with postnatal studies. The duplex LightCycler PCR test currently provides rapid and safe results. Copyright (C) 2001 John Wi ley & Sons, Ltd.