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

Abstract

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.

Peptide Microarray Analysis ofIn Silico-Predicted Epitopes for Serological Diagnosis of Toxoplasma gondii Infection in Humans

“…These institutions also provided 65 samples from serologically T. gondii-negative patients for the study. Detailed information about the serological status of patients with acute or latent T. gondii infection and T. gondii-seronegative patients was published previously by Maksimov et al (39). Sera collected from serologically T. gondii-positive (n ϭ 100) or -negative (n ϭ 75) forest workers were also used and were described in detail previously (39,40).…”

“…Sera collected from serologically T. gondii-positive (n ϭ 100) or -negative (n ϭ 75) forest workers were also used and were described in detail previously (39,40). The presence or absence of T. gondii-specific antibodies in the sera was confirmed by a latex agglutination test (LAT; see Table S1 in the supplemental material) and T. gondii surface antigen 1 (SAG1) immunoblot analysis (39).…”

“…Peptides were synthesized and printed on peptide microarray slides by JPT Peptide Technologies GmbH, Berlin, Germany, essentially as described by Maksimov et al (39). Human serum samples (60 l/well), diluted 1:200 in blocking solution, were incubated at 37°C for 1 h and washed seven times for 3 min each time with PBS-T (PBS [pH 7.2], 0.5% Tween 20) at room temperature.…”

“…Scanning and evaluation of microarray slides as well as data extraction were performed essentially as described previously (39,44,45).…”

“…To analyze the raw data (median of signal intensity) in GPR files, index values (IVs) were recovered for each peptide spot as the log 2 of the quotient of the medians of foreground and background values (39,44,45). Each serum sample was analyzed on a single block, with the peptides printed in triplicate on each block.…”