Significant Performance Variation Among PCR Systems in Diagnosing Congenital Toxoplasmosis in São Paulo, Brazil: Analysis of 467 Amniotic Fluid Samples

Abstract

INTRODUCTION 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. PATIENTS 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%). DISCUSSION 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. CONCLUSION 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.

Molecular diagnosis of cerebral toxoplasmosis: comparing markers that determine Toxoplasma gondii by PCR in peripheral blood from HIV-infected patients

“…These data are consistent with a recent Brazilian study conducted by Okay et al 21 In addition, previous studies have also shown high sensitivity and specifi city for markers designed to amplify different regions of the B1 gene in clinical samples. 13,18,19,25,27,29 Other studies conducted using European clinical samples aiming to compare sensitivities of both DNA regions have shown that those from repeat regions were more sensitive than those from the B1 gene.…”

“…[12][13][14][17][18][19][20] The majority of these studies aiming to compare sensitivities of both DNA regions were conducted in European clinical samples, but little research has been done on Brazilian samples. 21 These data, combined with certain limitations in laboratorial practices, such as the low parasite levels in some clinical samples, lead us to compare two molecular markers directed at the B1 gene and the 529-bp sequence for cerebral toxoplasmosis diagnosis.…”

A 10-Year Retrospective Comparison of Two Target Sequences, REP-529 and B1, for Toxoplasma gondii Detection by Quantitative PCR

“…However, a recent Brazilian study (23) reported a lower proportion of positive amniotic fluids with the REP-529 target than with B1 (36.5% and 87.3%, respectively), with only 23.8% of samples being positive with both targets. This unusual finding must be verified on other series of clinical samples from South America to check if atypical parasite strains circulating in this area might lack the REP-529 sequence or have mutations or modifications in the number of repetitions that might lead to a decreased sensitivity of this PCR target, which now appears to be the gold standard for European parasite strains.…”

Importance of Nonenteric Protozoan Infections in Immunocompromised People

“…In these patients, PCR applied to peripheral blood specimens could eliminate the requirement for invasive CNS biopsy techniques (429). A number of PCR assays have also been optimized for use on amniotic fluid for the diagnosis of congenital toxoplasmosis (188,404,409). As mentioned above in the section on pregnancy, primary Toxoplasma infections acquired prior to the commencement of a pregnancy pose little threat to the fetus.…”