Detection of toxoplasma gondii in amniotic fluid by a polymerase chain reaction/enzyme-linked immunosorbent assay (PCR/ELISA)
Genome-Wide Association Study
Nucleic Acid Amplification Techniques
Objectives: Maternal exposure to Toxoplasma gondü during pregnancy may result in congenital infection and serious sequela in the neonatal period or years after birth. However, there is little consensus about screening during pregnancy, and the tests used to establish a fetal diagnosis of toxoplasmosis are complex and time consuming. Prenatal diagnosis of congenital toxoplasmosis is based on ultrasonography, fetal blood sampling or inoculation of cultured cells or mice with amniotic fluid. Given the risk associated with fetal-blood sampling and the delay in obtaining definitive results with conventional parasitologic methods or ultrasound, better methods are needed. T. gondii has been diagnosed by PCR but detection of the amplified product by an ELISA has, to our knowledge, not been reported. ELISA detection has increased sensitivity over other methods. Study Design: Using human amniotic fluids that had been spiked with T. gondii, we established parameters for the amplification and detection of a fragment of the 35-fold repetitive Bl gcne.Thc 115-basc pair product, digoxigcnin-Iabelcd, was hybridized to a biotinylatcd internal probe and detected by ELISA. The hybridization validated the specificity of the amplified PCR product. The possible presence of PCR inhibitors in individual amniolic fluids was evaluated by amplifying a portion of the human beta aclin gene. Results: T. $ondu DNA was readily detected in amniotic fluids by our PCR/ELISA. PCR analysis of DNA extracted from various bacteria, yeast, and viruses using the 7". gondii primer pairs did not result in amplification of any crossrcacting DNA. Similarly, amniotic fluids obtained for genetic analysis were uniformly negative for T. gontlii. Conclusions: The PCR/ELISA will allow for the definitive diagnosis of congenital T. gondii infection in one day. This will reduce Ihe incidence of unnecessary pregnancy terminations based on uncertainty of fetal infection as well as allow the prompt initiation of antibiotic treatment to infected fetuses.