Abstract
BackgroundBecause some Toxoplasma gondii genotypes may be more virulent in pregnant women, discriminating between them appears valuable. Currently, the main genotyping method is based on single copy microsatellite markers, which limit direct genotyping from amniotic fluids (AFs) to samples with a high parasitic load. We investigated whether the multicopy gene B1 could type the parasite with a higher sensitivity. To estimate the amplifiable DNA present in AFs, we first compared three different PCR assays used for Toxoplasma infection diagnosis: the P30-PCR, targeting the single copy gene P30; the B1-PCR, targeting the repeated B1 gene; and RE-PCR, targeting the repeated element.ResultsOf the 1792 AFs analyzed between 2008 and 2011, 73 were RE-PCR positive. Of those, 49 (67.1%) were P30-PCR and B1-PCR positive, and 14 (19.2%) additional AFs were B1-PCR positive only.All 63 BI-positive AFs (France n = 49; overseas n = 14) could be genotyped based on an analysis of eight nucleotide polymorphisms (SNPs) located within the B1 gene. Following high-resolution melting (HRM) analysis, minisequencing was carried out for each of the eight SNPs. DNA from six reference strains was included in the study, and AFs were assigned to one of the three major lineages (Types I, II, and III). In total, 26 genotypes were observed, and the hierarchical clustering distinguished two clades in lineages II (IIa, n = 30 and IIb, n = 4) and III (IIIa n = 23 and IIIb n = 6). There was an overrepresentation of overseas isolates in Clade IIb (4/4, 100%) and Clade IIIa (8/22; 36.4%) (p <0.0001), whereas medical interruption and fetal death were overrepresented in Clade IIb (2/4, 50%) and Clade IIIa (4/23, 17.4%) (p = 0.049).ConclusionsAlthough the current genotyping system cannot pretend to replace multilocus typing, we clearly show that targeting the multicopy B1 gene yields a genotyping capacity of AFs around 20% better than when single copy targets are used. The present genotyping method also allows clear identification of genotypes of potential higher virulence.
Highlights
Because some Toxoplasma gondii genotypes may be more virulent in pregnant women, discriminating between them appears valuable
We recently investigated the possibility of genotyping T. gondii using the polymorphism of the repeated B1 gene with high-resolution melting (HRM) analysis and minisequencing (SNaPshot) using reference strains of different types [10]
According to the repeated element (RE)-PCR assay, which is used for routine testing in our diagnostic laboratory [16], T. gondii DNA was detected in 82/1792 (4.6%) amniotic fluids (AFs)
Summary
Because some Toxoplasma gondii genotypes may be more virulent in pregnant women, discriminating between them appears valuable. The main genotyping method is based on single copy microsatellite markers, which limit direct genotyping from amniotic fluids (AFs) to samples with a high parasitic load. To estimate the amplifiable DNA present in AFs, we first compared three different PCR assays used for Toxoplasma infection diagnosis: the P30-PCR, targeting the single copy gene P30; the B1-PCR, targeting the repeated B1 gene; and RE-PCR, targeting the repeated element. Infection by the protozoan parasite Toxoplasma gondii can yield symptoms ranging from asymptomatic infections to miscarriages or meningoencephalitis in immunocompromised patients. Genotyping has revealed genetic diversity in this worldwide parasite, and three main lineages (Types I, II, and III) [2], grouped into 15 haplogroups in six major clades, have been described [3]. The need for a simple genotyping system, effective in clinical specimens, is clear
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