Genotyping of Toxoplasma gondii by Multiplex PCR and Peptide-Based Serological Testing of Samples from Infants in Poland Diagnosed with Congenital Toxoplasmosis

Abstract

Toxoplasma gondii has a clonal population genetic structure with three (I, II, and III) lineages that predominate in North America and Europe. Type II strains cause most cases of symptomatic human infections in France and the United States, although few other regions have been adequately sampled. Here we determined the parasite genotype in amniotic fluid and cerebrospinal fluid samples from congenital toxoplasmosis cases in Poland. Nineteen confirmed congenital cases of toxoplasmosis were analyzed, including both severe and asymptomatic cases. The genotype of parasite strains causing congenital infection was determined by direct PCR amplification and restriction fragment length polymorphism analysis. Nested multiplex PCR analysis was used to type four independent polymorphic markers. The sensitivity of multiplex nested PCR was >25 parasites/ml in amniotic fluid and cerebral spinal fluid samples. Parasite DNA was successfully amplified in 9 of 19 samples (eight severely affected and one asymptomatic fetus). Only genotype II parasites were identified as the source of T. gondii infection based on restriction fragment length polymorphism analysis. Strains causing congenital infections were also typed indirectly based on detection of antibodies to strain-specific peptides. Serotyping indicated that 12 of 15 cases tested were caused by type II strains and these positives included both symptomatic and asymptomatic infections. Overall, the combined analysis indicated that 14 of the cases were caused by type II strains. Our results are consistent with the hypothesis that parasite burden is associated with severity of congenital toxoplasmosis and indicate that serological testing provides a promising method for genotypic analysis of toxoplasmosis.

Molecular Detection and Genotyping of Toxoplasma gondii from Clinical Samples

“…The greatest limitation in genotyping of isolates from clinical samples is the small number of parasites in original material; hence the amount of extracted T. gondii DNA is often also small. This problem can be partially eliminated by enriching the sample by bioassay or cell culture, but even the most sensitive molecular methods, such as a multiplex nested PCR, have a threshold of 50 and 25 parasites/mL, respectively (Khan et al, 2005;Nowakowska et al, 2006). The PCR-RFLP protocol by which genotyping was performed in our study has a sensitivity of approximately 170 parasites/mL, which is probably the major reason for the small number of successful genotypizations.…”

“…Therefore, there are cases when amplification of all markers in each sample is not successful, as it can be observed in studies performed in the United States and Poland, where PCR-RFLP analysis was carried out also using four genetic markers SAG2, SAG3, BTUB and GRA6 (Khan et al, 2005;Nowakowska et al, 2006). Using these genetic markers, it was possible to discriminate types I, II and III, but also strains that have a genotype with two allele types at the same locus.…”

“…One French study has shown that of the 86 isolates from cases of suspected and confirmed congenital toxoplasmosis 85% were of type II (Ajzenberg et al, 2002). A predominance of the same type was indicated in Poland, where genotyping was also performed in samples originating from clinical cases of congenital toxoplasmosis (Nowakowska et al, 2006). In South-East Europe the first strain genotyped was isolated from a case of congenital toxoplasmosis in Serbia, and was also designated as type II (Djurković-Djaković et al, 2006).…”

Is congenital toxoplasmosis still an important clinical problem?

“…This could be explained by the increased permeability of placental barriers throughout the pregnancy. On the contrary, the earlier the infection, the worst the outcomes are to be expected, for instance, it can lead to miscarriages or serious neurological defects [2,6,11]. In severe to moderate cases, the classical triad of CT is occasionally seen at birth, including; retinochoroiditis, intracranial calcifications and hydrocephalus in 80%, 40% and 20%, respectively [12].…”

Experimental Models of Ocular Toxoplasmosis

“…Studies of Howe and Sibley, in 1995 and Belfort-Neto and collaborators in 2007 correlate type II with an important role in acquired infection, while type I clonal lineage may be responsible for the congenital toxoplasmosis, even so type I and atypical strains may play a role in acquired infection [14]. For instance, type III has been reported only 9% of toxoplasmosis patients in France and United States [17]. However, even with few information relating clonal type with virulence and frequency, just an insufficient part is known, needing more studies in this area.…”