Fig 1.
Chagas-Flow ATE-IgG2a performance to discriminate serological reactivity during early and late T. cruzi infections.
(A) Overall reactivity profile of sera samples from early (n = 110) and late (n = 200) T. cruzi infections and non-infected hosts (n = 10). The IgG2a reactivity was evaluated along the titration curve with each target-antigens amastigote (AMA = green), trypomastigote (TRYPO = red) and epimastigote (EPI = yellow) from TcI, TcVI and TcII genotypes of T. cruzi. The results are displayed in radar charts and expressed as mean of percentage of positive fluorescent parasites (PPFP). (B) Titration curves were assessed at eight serum dilutions (1:500 to 1:64,000) using target-antigens AI, AVI and AII; TI, TVI and TII along with EI, EVI and EI at early (triangle) and late (square) stages of T. cruzi infection as compared to non-infected hosts (circle). (C) The reactivity amongst groups was compared for pre-selected pairs of attributes, including: AI 500; AVI 500; AII 500; TI 16,000; TVI 4,000; TII 32,000; EI 2,000; EVI 500 and EII 500. The results are expressed as median PPFP values in box plot format with the outliers underscored by shaded dots. Comparative analyses were performed by the Kruskal-Wallis/Dunn’s post test for multi-group comparisons. Significant differences were considered at p<0.05 and highlighted by connecting lines. The light gray background underscores the pair of attributes (“target antigen and serum dilution”) with the higher performance to discriminate early and late T. cruzi infections. (D) Decision trees were constructed using sets of attributes (“target-antigen and serum dilution/cut-off”) to create algorithms (root and branches) to classify individual samples at early and late T. cruzi infections. Global accuracy and leave-one-out-cross-validation-LOOCV are provided in the Figure.
Fig 2.
Overall Chagas-Flow ATE-IgG2a reactivity at early and late stages of single and dual T. cruzi infections.
(A) Panoramic reactivity profile of sera samples at early stage of single (Triangle, n = 50) and dual (Cross, n = 60) as well as at late stage of single (Square, n = 93) and dual (Asterisk, n = 107) T. cruzi infections. The IgG2a reactivity was evaluated along the titration curve with each target-antigens amastigote (AMA = green), trypomastigote (TRYPO = red) and epimastigote (EPI = yellow) from TcI, TcVI and TcII genotypes of T. cruzi. The results are shown in radar charts and expressed as mean of percentage of positive fluorescent parasites (PPFP). (B) Titration curves at eight serum dilutions (1:500 to 1:64,000) using target-antigens AI, AVI and AII; TI, TVI and TII and EI, EVI and EII were assessed at early and late stages of single and dual T. cruzi infections.
Fig 3.
Selection of attributes for Chagas-Flow ATE-IgG2a to discriminate single and dual T. cruzi infections at early and late stages.
(A) Comparative analysis of IgG2a reactivity at early stage of single (n = 50) and dual (n = 60) T. cruzi infections, using pre-selected pairs of attributes, including: AI 1,000; AVI 8,000; AII 2,000; TI 2,000; TVI 2,000; TII 8,000; EI 2,000; EVI 2,000 and EII 1,000. (B) Comparative analysis of IgG2a reactivity at late stage of single (n = 93) and dual (n = 107) T. cruzi infections, using pre-selected pairs of attributes, including: AI 500; AVI 500; AII 32,000; TI 8,000; TVI 4,000; TII 16,000; EI 4,000; EVI 2,000 and EII 4,000. The results are expressed as median of PPFP values in box plot format with the outliers showed by shaded dots. Comparative analyses were performed by the Kruskal-Wallis/Dunn’s post test. Significant differences were considered at p<0.05 and highlighted by connecting lines. The light gray background highlights the pairs of attributes (“target antigen and serum dilution”) with the higher performance to discriminate single and dual T. cruzi infections at early and late stages. Decision trees were constructed using the sets of attributes (“target-antigen and serum dilution/cut-off”) to create algorithms (root and branches) to classify individual samples from single and dual T. cruzi infections at (C) early and (D) late stages. Global accuracy and leave-one-out-cross-validation-LOOCV are provided in the Figure.
Fig 4.
Genotype-specific profile of Chagas-Flow ATE-IgG2a reactivity at early stage of single and dual T. cruzi infections.
(A) Overall reactivity profile of sera samples at early stage of single (TcI/COL, n = 16; TcVI/CL, n = 15 and TcII/Y, n = 19) and dual (TcI/COL+TcVI/CL, n = 16; TcVI/CL+TcII/Y, n = 24 and TcI/COL+TcII/Y, n = 20) T. cruzi infections with distinct genotypes. The IgG2a reactivity was evaluated along the titration curve with each target-antigens amastigote (AMA = green), trypomastigote (TRYPO = red) and epimastigote (EPI = yellow) from TcI, TcVI and TcII genotypes of T. cruzi. The results are shown on radar charts and expressed as mean of percentage of positive fluorescent parasites (PPFP). (B) Titration curves were assessed at eight serum dilutions (1:500 to 1:64,000) using target-antigens AI, AVI and AII; TI, TVI and TII along with EI, EVI and EI at early stage of single [TcI/COL (triangle), TcVI/CL (square), TcII/Y (circle)] and dual [TcI/COL+TcVI/CL (cross), TcVI/CL+TcII/Y (dash), TcI/COL+TcII/Y (asterisk)] T. cruzi infections.
Fig 5.
Selection of attributes for genotype-specific Chagas-Flow ATE-IgG2a at early stage of single and dual T. cruzi infections.
(A) Comparative analysis of IgG2a reactivity at early stage of single infection with distinct T. cruzi genotypes (TcI/COL, n = 16; TcVI/CL, n = 15 and TcII/Y, n = 19) using pre-selected pairs of attributes, including: AI 1,000; AVI 500; AII 500; TI 4,000; TVI 500; TII 1,000; EI 1,000; EVI 500 and EII 500. (B) Comparative analysis of IgG2a reactivity at early stage of dual infection with distinct T. cruzi genotypes (TcI/COL+TcVI/CL, n = 16; TcVI/CL+TcII/Y, n = 24 and TcI/COL+TcII/Y, n = 20) using pre-selected pairs of attributes, including: AI 500; AVI 1,000; AII 2,000; TI 16,000; TVI 500; TII 8,000; EI 2,000; EVI 2,000 and EII 2,000. The results are expressed as median PPFP values in box plot format with the outliers showed by shaded dots. Comparative analyses were performed by the Kruskal-Wallis/Dunn’s post test. Significant differences were considered at p<0.05 and highlighted by connecting lines. The light gray background highlights the pairs of attributes (“target antigen and serum dilution”) with the higher performance for the genotype-specific diagnosis at early stage of single and dual T. cruzi infections. Decision trees were constructed using the sets of attributes (“target-antigen and serum dilution/cut-off”) to create algorithms (root and branches) to classify at early stage, individual samples from (C) single and (D) dual T. cruzi infections. Global accuracy and leave-one-out-cross-validation-LOOCV are provided in the Figure.
Fig 6.
Genotype-specific profile of Chagas-Flow ATE-IgG2a reactivity at late stage of single and dual T. cruzi infections.
(A) Overall reactivity profile of sera samples at late stage of single (TcI/COL, n = 29; TcVI/CL, n = 29 and TcII/Y, n = 35) and dual (TcI/COL+TcVI/CL, n = 28; TcVI/CL+TcII/Y, n = 43 and TcI/COL+TcII/Y, n = 36) T. cruzi infections with distinct genotypes. The IgG2a reactivity was evaluated along the titration curve with each target-antigens amastigote (AMA = green), trypomastigote (TRYPO = red) and epimastigote (EPI = yellow) from TcI, TcVI and TcII genotypes of T. cruzi. The results are shown on radar charts and expressed as mean of percentage of positive fluorescent parasites (PPFP). (B) Titration curves were assessed at eight serum dilutions (1:500 to 1:64,000) using target-antigens AI, AVI and AII; TI, TVI and TII along with EI, EVI and EI at late stage of single [TcI/COL (triangle), TcVI/CL (square), TcII/Y (circle)] and dual [TcI/COL+TcVI/CL (cross), TcVI/CL+TcII/Y (dash), TcI/COL+TcII/Y (asterisk)] T. cruzi infections.
Fig 7.
Selection of attributes for genotype-specific Chagas-Flow ATE-IgG2a at late stage of single and dual T. cruzi infections.
(A) Comparative analysis of IgG2a reactivity at late stage of single infection with distinct T. cruzi genotypes (TcI/COL, n = 29; TcVI/CL, n = 29 and TcII/Y, n = 35) using pre-selected pairs of attributes, including: AI 2,000; AVI 1,000; AII 1,000; TI 4,000; TVI 1,000; TII 16,000; EI 2,000; EVI 1,000 and EII 4,000. (B) Comparative analysis of IgG2a reactivity at late stage of dual infection with distinct T. cruzi genotypes (TcI/COL+TcVI/CL, n = 28; TcVI/CL+TcII/Y, n = 43 and TcI/COL+TcII/Y, n = 36) using pre-selected pairs of attributes, including: AI 2,000; AVI 500; AII 1,000; TI 64,000; TVI 16,000; TII 64,000; EI 4,000; EVI 16,000 and EII 4,000. The results are expressed as median PPFP values in box plot format with the outliers showed by shaded dots. Comparative analyses were performed by the Kruskal-Wallis/Dunn’s post test. Significant differences were considered at p<0.05 and highlighted by connecting lines. The light gray background highlights the pairs of attributes (“target antigen and serum dilution”) with the higher performance for the genotype-specific diagnosis at late stage of single and dual T. cruzi infections. Decision trees were constructed using the sets of attributes (“target-antigen and serum dilution/cut-off”) to create algorithms (root and branches) to classify at late stage, individual samples from (C) single and (D) dual T. cruzi infections. Global accuracy and leave-one-out-cross-validation-LOOCV are provided in the Figure.
Fig 8.
Overall performance of Chagas-Flow ATE-IgG2a for differential diagnosis of experimental T. cruzi infection.
(A) Reactivity boards were employed to display the overall profile of Chagas-Flow ATE-IgG2a applied to the differential diagnosis of T. cruzi infection at early (left diagram) and late (right diagram), using the selected sets of attributes (“target-antigen and serum dilution/cut-off”) determined by the decision trees. (B) Sequential algorithms were applied to yield the “classified infection” status to each individual sample, referred as: “a” for early vs late; “b” for single vs dual infections at early stage; “c” for single vs dual infections at late stage; “d” for genotype-specific diagnosis at early stage of single infection; “e” for genotype-specific diagnosis at early stage of dual infection; “f” for genotype-specific diagnosis at late stage of single infection and “g” for genotype-specific diagnosis at late stage of dual infection. A color key was provided to identify early (yellow), late (brown), single (light gray), dual (dark gray), TcI/COL (red), TcVI/CL (green), TcII/Y (dark blue), TcI/COL+TcVI/CL (orange), TcVI/CL+TcII/Y (light blue) and TcI/COL+TcII/Y (pink). (C) Scatter graphs showed correspondence between each pair of “true” vs “classified” infection indicated by connecting lines and the percentage of agreement for “Stage & Type & Genotype”; “Type & Genotype” and “Type” of infection. (D) Discriminant analyses of combined Chagas-Flow ATE-IgG2a for genotype-specific diagnosis including T. cruzi single infection (COL, CL and Y) and dual infection (COL+CL, CL+Y and COL+Y). The global accuracy is provided in the Figure.