Analysis of Clonal Type-Specific Antibody Reactions in Toxoplasma gondii Seropositive Humans from Germany by Peptide-Microarray

BACKGROUND: Different clonal types of Toxoplasma gondii are thought to be associated with distinct clinical manifestations of infections. Serotyping is a novel technique which may allow to determine the clonal type of T. gondii humans are infected with and to extend typing studies to larger populations which include infected but non-diseased individuals. METHODOLOGY: A peptide-microarray test for T. gondii serotyping was established with 54 previously published synthetic peptides, which mimic clonal type-specific epitopes. The test was applied to human sera (n = 174) collected from individuals with an acute T. gondii infection (n = 21), a latent T. gondii infection (n = 53) and from T. gondii-seropositive forest workers (n = 100). FINDINGS: The majority (n = 124; 71%) of all T. gondii seropositive human sera showed reactions against synthetic peptides with sequences specific for clonal type II (type II peptides). Type I and type III peptides were recognized by 42% (n = 73) or 16% (n = 28) of the human sera, respectively, while type II-III, type I-III or type I-II peptides were recognized by 49% (n = 85), 36% (n = 62) or 14% (n = 25) of the sera, respectively. Highest reaction intensities were observed with synthetic peptides mimicking type II-specific epitopes. A proportion of the sera (n = 22; 13%) showed no reaction with type-specific peptides. Individuals with acute toxoplasmosis reacted with a statistically significantly higher number of peptides as compared to individuals with latent T. gondii infection or seropositive forest workers. CONCLUSIONS: Type II-specific reactions were overrepresented and higher in intensity in the study population, which was in accord with genotyping studies on T. gondii oocysts previously conducted in the same area. There were also individuals with type I- or type III-specific reactions. Well-characterized reference sera and further specific peptide markers are needed to establish and to perform future serotyping approaches with higher resolution

Strongest reaction intensities were recorded for clonal type II specific peptides.

<p>To evaluate the intensities (MSIVs) by which single peptides as well as peptide cohorts (I, II, III, I–II, I–III, or II–III) were recognized by <i>T. gondii</i> seropositive patient and volunteer groups, ANOVA and the Least Significant Difference (LSD)-Post-Hoc-Test were performed. Whiskers in barplots represent 95% confidence intervals of the means of MSIVs. The differences between the means of MSIVs for single peptides or peptide cohorts within tested groups were regarded as statistically significant, when the differences were equal or higher than the LSD values. Different letters above the whiskers indicate significant differences between the mean intensities in the Post-Hoc-LSD test. Means of MSIVs for each peptide cohort are presented in (A) for the acutely infected patient group (LSD>0.36, p-value<0.05); in (C) for the latently infected patient group (LSD>0.16, p-value<0.05); and in (E) for the seropositive volunteer group (LSD>0.10, p-value<0.05). Means of MSIVs for each single peptide are presented in (B) within the acutely infected patient group (LSD>0.67, p-value<0.05); in (D) within the latently infected patient group (LSD>0.36, p-value<0.05); and in (F) within the seropositive volunteer group (LSD>0.24, p-value<0.05).</p

Proportion of human sera showing peptide reactions compatible with <i>T. gondii</i> infections by clonal types I, II, or III.

<p>Reactions are sorted according to their compatibility with infections of <i>T. gondii</i> of the clonal type I, II, or III.</p>*<p>Data resolved for seropositive patients with acute toxoplasmosis (AP), patients with latent toxoplasmosis (LP) and seropositive volunteers (V).</p

Clonal type-specific anti-peptide reactivity of <i>T. gondii</i> positive humans.

<p>Reactions are sorted according to the specificities of peptides.</p

The number of recognized peptides is associated with the titre in LAT (Latex Agglutination Test).

<p>Wilcoxon rank test analysis of the <i>T. gondii</i> LAT titre distribution within groups of human sera revealed significantly lower LAT titres in sera from volunteers than in sera from latently or in acutely infected individuals (p-value<0.001). No significant differences were observed between sera from acutely and latently infected individuals (A). The association between LAT titre and number of recognized peptides was characterized by an R² value of 0.16 (p-value<0.001) (B).</p