title: Significant performance variation among PCR systems in diagnosing congenital toxoplasmosis in São Paulo, Brazil: analysis of 467 amniotic fluid samples.
abstract: BACKGROUND
  Performance variation among PCR systems in detecting Toxoplasma gondii has been extensively reported and associated with target genes, primer composition, amplification parameters, treatment during pregnancy, host genetic susceptibility and genotypes of different parasites according to geographical characteristics.
  
  
  METHODS
  A total of 467 amniotic fluid samples from T. gondii IgM- and IgG-positive Brazilian pregnant women being treated for 1 to 6 weeks at the time of amniocentesis (gestational ages of 14 to 25 weeks).
  
  
  METHODS
  One nested-B1-PCR and three one-round amplification systems targeted to rDNA, AF146527 and the B1 gene were employed.
  
  
  RESULTS
  Of the 467 samples, 189 (40.47%) were positive for one-round amplifications: 120 (63.49%) for the B1 gene, 24 (12.69%) for AF146527, 45 (23.80%) for both AF146527 and the B1 gene, and none for rDNA. Fifty previously negative one-round PCR samples were chosen by computer-assisted randomization analysis and re-tested (nested-B1-PCR), during which nine additional cases were detected (9/50 or 18%).
  
  
  CONCLUSIONS
  The B1 gene PCR was far more sensitive than the AF146527 PCR, and the rDNA PCR was the least effective even though the rDNA had the most repetitive sequence. Considering that the four amplification systems were equally affected by treatment, that the amplification conditions were optimized for the target genes and that most of the primers have already been reported, it is plausible that the striking differences found among PCR performances could be associated with genetic diversity in patients and/or with different Toxoplasma gondii genotypes occurring in Brazil.
  
  
  CONCLUSIONS
  The use of PCR for the diagnosis of fetal Toxoplasma infections in Brazil should be targeted to the B1 gene when only one gene can be amplified, preferably by nested amplification with primers B22/B23.
authors.affiliation: Laboratory of Medical Investigation, Instituto da Criança, Faculdade de Medicina da Universidade de São Paulo - São Paulo/SP, Brazil. thelma.okay@icr.usp.br
authors.forename: Thelma Suely
authors.initials: TS
authors.lastname: Okay
authors.forename: Lidia
authors.initials: L
authors.lastname: Yamamoto
authors.forename: Léa Campos
authors.initials: LC
authors.lastname: Oliveira
authors.forename: Erika Regina
authors.initials: ER
authors.lastname: Manuli
authors.forename: Heitor Franco de
authors.initials: HF
authors.lastname: Andrade Junior
authors.forename: Gilda Maria Barbaro
authors.initials: GM
authors.lastname: Del Negro