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<p>(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) <i>T</i>. <i>cruzi</i> 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 <i>T</i>. <i>cruzi</i>. 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)] <i>T</i>. <i>cruzi</i> infections.</p

Selection of attributes for genotype-specific Chagas-Flow ATE-IgG2a at late stage of single and dual <i>T</i>. <i>cruzi</i> infections.

<p>(A) Comparative analysis of IgG2a reactivity at late stage of single infection with distinct <i>T</i>. <i>cruzi</i> 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 <i>T</i>. <i>cruzi</i> 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 <i>T</i>. <i>cruzi</i> 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 <i>T</i>. <i>cruzi</i> infections. Global accuracy and leave-one-out-cross-validation-LOOCV are provided in the Figure.</p

Overall Chagas-Flow ATE-IgG2a reactivity at early and late stages of single and dual <i>T</i>. <i>cruzi</i> infections.

<p>(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) <i>T</i>. <i>cruzi</i> 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 <i>T</i>. <i>cruzi</i>. 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 <i>T</i>. <i>cruzi</i> infections.</p

Selection of attributes for Chagas-Flow ATE-IgG2a to discriminate single and dual <i>T</i>. <i>cruzi</i> infections at early and late stages.

<p>(A) Comparative analysis of IgG2a reactivity at early stage of single (n = 50) and dual (n = 60) <i>T</i>. <i>cruzi</i> 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) <i>T</i>. <i>cruzi</i> 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 <i>T</i>. <i>cruzi</i> 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 <i>T</i>. <i>cruzi</i> infections at (C) early and (D) late stages. Global accuracy and leave-one-out-cross-validation-LOOCV are provided in the Figure.</p

Selection of attributes for genotype-specific Chagas-Flow ATE-IgG2a at early stage of single and dual <i>T</i>. <i>cruzi</i> infections.

<p>(A) Comparative analysis of IgG2a reactivity at early stage of single infection with distinct <i>T</i>. <i>cruzi</i> 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 <i>T</i>. <i>cruzi</i> 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 <i>T</i>. <i>cruzi</i> 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 <i>T</i>. <i>cruzi</i> infections. Global accuracy and leave-one-out-cross-validation-LOOCV are provided in the Figure.</p

Performance of TcI/TcVI/TcII Chagas-Flow ATE-IgG2a for universal and genotype-specific serodiagnosis of <i>Trypanosoma cruzi</i> infection

<div><p>Distinct <i>Trypanosoma cruzi</i> genotypes have been considered relevant for patient management and therapeutic response of Chagas disease. However, typing strategies for genotype-specific serodiagnosis of Chagas disease are still unavailable and requires standardization for practical application. In this study, an innovative TcI/TcVI/TcII Chagas Flow ATE-IgG2a technique was developed with applicability for universal and genotype-specific diagnosis of <i>T</i>. <i>cruzi</i> infection. For this purpose, the reactivity of serum samples (percentage of positive fluorescent parasites-PPFP) obtained from mice chronically infected with TcI/Colombiana, TcVI/CL or TcII/Y strain as well as non-infected controls were determined using amastigote-AMA, trypomastigote-TRYPO and epimastigote-EPI in parallel batches of TcI, TcVI and TcII target antigens. Data demonstrated that “α-TcII-TRYPO/1:500, cut-off/PPFP = 20%” presented an excellent performance for universal diagnosis of <i>T</i>. <i>cruzi</i> infection (AUC = 1.0, Se and Sp = 100%). The combined set of attributes “α-TcI-TRYPO/1:4,000, cut-off/PPFP = 50%”, “α-TcII-AMA/1:1,000, cut-off/PPFP = 40%” and “α-TcVI-EPI/1:1,000, cut-off/PPFP = 45%” showed good performance to segregate infections with TcI/Colombiana, TcVI/CL or TcII/Y strain. Overall, hosts infected with TcI/Colombiana and TcII/Y strains displayed opposite patterns of reactivity with “α-TcI TRYPO” and “α-TcII AMA”. Hosts infected with TcVI/CL strain showed a typical interweaved distribution pattern. The method presented a good performance for genotype-specific diagnosis, with global accuracy of 69% when the population/prototype scenario include TcI, TcVI and TcII infections and 94% when comprise only TcI and TcII infections. This study also proposes a receiver operating reactivity panel, providing a feasible tool to classify serum samples from hosts infected with distinct <i>T</i>. <i>cruzi</i> genotypes, supporting the potential of this method for universal and genotype-specific diagnosis of <i>T</i>. <i>cruzi</i> infection.</p></div

Overall reactivity profile of TcI/TcVI/TcII Chagas-Flow ATE-IgG2a for universal and genotypic-specific diagnosis of <i>T</i>. <i>cruzi</i> infection.

<p>The Chagas-Flow ATE-IgG2a reactivity was determined for sera samples from <i>T</i>. <i>cruzi</i>-infected mice, including TcI/TcVI/TcII genotype-representative strains, including: TcI/Colombiana strain (black dotted frame, n = 29), TcVI/CL strain (light gray dotted frame, n = 29) and TcII/Y strain (dark gray dotted frame, n = 35) as well as non-infected mice (white dotted frame, n = 10). Genotype-specific IgG2a reactivity to each target-antigen (amastigote-AMA, trypomastigote-TRYPO and epimastigote-EPI) from <i>T</i>. <i>cruzi</i> genotype I (left panels), genotype VI (middle panels) and genotype II (right panels) was assessed at eight serum dilutions (1:500 to 1:64,000). The results are expressed as the percentage of positive fluorescent parasites (PPFP), using the box plot format, stretching from min to max values with outliers represented by gray-shaded dots and the box defining the 25^th and 75^th percentile and the median value (line across the box). Comparative analyses were performed by the Kruskal-Wallis followed by Dunn’s post test for multi-group comparisons. Significant differences were considered at p<0.05. The light gray continuous rectangle selects the pair of attributes (“target antigen/serum dilution”) with the most consistent ability to discriminate non-infected mice from all <i>T</i>. <i>cruzi</i>-infected hosts (Colombiana, CL and Y strains). Therefore, these features (anti-TcII TRYPO reactivity at 1:500) were selected for universal diagnosis of <i>T</i>. <i>cruzi</i> infection. The dark gray dotted frame select the pair of attributes “target antigen/serum dilution” with the most promising perspective to distinguish the reactivity of sera samples amongst host infected with Colombiana, CL or Y <i>T</i>. <i>cruzi</i> strains. Therefore, these features (anti-TcII AMA reactivity at 1:1,000; anti-TcI TRYPO reactivity at 1:4,000 and anti-TcVI EPI reactivity at 1:1,000) were selected for genotype-specific diagnosis of <i>T</i>. <i>cruzi</i> infection.</p

Criteria to define universal and genotype-specific diagnosis of <i>T</i>. <i>cruzi</i> infection by TcI/TcVI/TcII Chagas-Flow ATE-IgG2a in two population/prototype scenarios.

<p>(A) Reactivity boards were built to provide a panoramic snapshot of TcI/TcVI/TcII Chagas-Flow ATE-IgG2a applied to the universal and genotype-specific diagnosis of <i>T</i>. <i>cruzi</i> infection. Data mining approaches were used to pre-select the target-antigens and specific cut-off edges to define positive results for universal diagnosis purpose (TcII TRYPO, PPFP>20%) and genotype-specific diagnosis criteria (TcI TRYPO, PPFP>50%; TcII AMA, PPFP>40% and TcVI EPI, PPFP>45%) based on the differential positive reactivity of sera samples (TcI/Colombiana strain = black rectangle, TcVI/CL strain = light gray rectangle and TcII/Y strain = dark gray rectangle) from negative reactivity (white rectangle) observed for <i>T</i>. <i>cruzi</i> infected hosts and non-infected mice. The pre-selected sera dilutions defined by decision tree analysis are underscored by dotted rectangles and include TcII TRYPO PPFP>20% at 1:500 for universal diagnosis and the set of attributes (TcI-TRYPO/PPFP>50%/4,000 followed by TcII-AMA/PPFP>40%/1,000 and TcVI-EPI/PPFP>45%/1,000) for genotype-specific diagnosis criteria. Reactivity panels were constructed to define the diagnosis conclusion when applying TcI/TcVI/TcII Chagas-Flow ATE-IgG2a for (B) universal diagnosis of <i>T</i>. <i>cruzi</i> infection and genotype-specific diagnosis in a population/prototype including TcI/Colombiana strain (COL), TcVI/CL strain (CL) or TcII/Y (Y) strain or (C) including TcI/Colombiana strain (COL) or TcII/Y strain (Y).</p

Outline of TcI/TcVI/TcII Chagas-Flow ATE-IgG2a for serodiagnosis of <i>Trypanosoma cruzi</i> infection.

<p>(A) The experimental procedure display a schematic representation of genotype-specific <i>T</i>. <i>cruzi</i> ATE-parasite Mix Platforms using TcI (Colombiana strain) = black bar, TcVI (CL strain) = light gray bar and TcII (Y strain) = dark gray bar antigens in separate batches. (B) Representative gating strategies used to select the target antigens (amastigote-AMA, trypomastigote-TRYPO and epimastigote-EPI) on each Chagas-Flow ATE-IgG2a platform and the histograms employed to quantify the genotype-specific anti-<i>T</i>. <i>cruzi</i> IgG2a reactivity, expressed by the percentage of positive fluorescent parasites (PPFP), based on the positivity limit (PPFP<2%), set based on the internal control.</p

Performance of TcI/TcVI/TcII Chagas-Flow ATE-IgG2a for universal diagnosis of <i>T</i>. <i>cruzi</i> infection.

<p>(A) The anti-TcII TRYPO reactivity at 1:500, pre-selected as attributes pairs for universal <i>T</i>. <i>cruzi</i> infection diagnosis, were compared by Kruskal-Wallis followed by Dunn’s post test for multi-group comparisons and significant differences at *p <0.05, **p<0,001 and ***p<0,0001, highlighted by connecting lines. Data are expressed as median PPFP values for non-infected mice (white bar) and <i>T</i>. <i>cruzi</i>-infected hosts (TcI/Colombiana strain = black bar, TcVI/CL strain = light gray bar and TcII/Y strain = dark gray bar). The similarity amongst the anti-TcII TRYPO IgG2a reactivity at 1:500 observed for the three <i>T</i>. <i>cruzi</i> infected groups (Colombiana + CL + Y strains) allows the establishment of a single group referred to as <i>T</i>. <i>cruzi</i> infected hosts (n = 93) and the performance of the TcI/TcVI/TcII Chagas-Flow ATE-IgG2a in the universal diagnosis of <i>T</i>. <i>cruzi</i> infection carried out as compared to a group of non-infected mice (NI, n = 10). (B) ROC-curve analysis was applied to define the appropriated cut-off to discriminate the PPFP values from NI and <i>T</i>. <i>cruzi</i>-infected host (Colombiana + CL + Y strains). Additional performance indices were also calculated and provided in the figure, including the area under the curve (AUC), defined as global accuracy, the sensitivity (Se) and the specificity (Sp). (C) Representative scatter plot illustrates the ability of the selected set of attributes (“target-antigen/serum dilution/cut-off”) to discriminate the reactivity of the sera from non-infected (NI) and <i>T</i>. <i>cruzi</i>-infected hosts (Colombiana+CL+Y). The dotted line represented the cut-off of PPFP = 20% defined by the ROC-curve analysis. (D) TG-ROC analysis was also performed to confirm the cut-off selection at higher “Se” and “Sp”, highlighted by dark gray dotted frame.</p