Immunodepletion assay showing specific IgG antibody recognition of the synthetic peptides with known reactivity to Cathepsin L-like.

<p>Pools of sera (n = 10) from the different groups were depleted with peptide-1 (CT, control group; CD, Chagas disease; CL, cutaneous leishmaniasis; ML, mucosal leishmaniasis; VL, visceral leishmaniasis; CVL, canine visceral leishmaniasis). The mean antibody OD values are shown on the <i>y</i>-axis, and the error bars indicate the standard deviation. Significant differences are indicated on the graphs (*<i>p</i><0.05; **<i>p</i><0.01; ***<i>p</i><0.001).</p

Diagnostic performance of <i>r</i>CatL, peptide-1, SLbA and the EIE-LVC kit using ROC curves. Data validation and agreement was confirmed using a kappa index.

a<p>The kappa index was calculated using all samples presented in this work for TL (CT + CD + CL + ML. n = 135), VL (CT + CD + VL. n = 125) and CVL (CT + CD + CVL. n = 75).</p>b<p>Agreement was calculated using parasitological assays as the gold standard.</p><p>*<i>Cut-off</i> obtained by ROC curve.</p>#<p><i>Cut-off</i> suggested by the manufacturer.</p><p>Abbreviations: AUC: area under curve; CI: confidence interval; TP: true positive; TN: true negative; FP: false positive; FN: false negative; κ: kappa index; NA: not applicable.</p><p>Diagnostic performance of <i>r</i>CatL, peptide-1, SLbA and the EIE-LVC kit using ROC curves. Data validation and agreement was confirmed using a kappa index.</p

Expression and purification of recombinant Cathepsin L-like protein.

<p>Protein samples were separated by 12.5% SDS-PAGE gel electrophoresis. (<b>A</b>) Molecular weight standard, (<b>B</b>) lysate of culture before and (<b>C</b>) after induction with IPTG and (<b>D</b>) recombinant Cathepsin L-like protein (MW 47.6 KDa) purified by gel filtration.</p

Comparison of ROC curves obtained from <i>r</i>CatL, Peptide-1 and SLbA.

<p>The ROC curves were used to determine the ELISA cut-off, sensitivity, specificity and AUC. In case of SLbA for CVL diagnosis (EIE-LVC Kit), ROC curve is not shown (not applicable) in this graph because the cut-off was determined according to the recommendations by the manufacturer (twice the average of the negative control included in kit).</p

Diagnostic performance of <i>r</i>CatL, peptide-1, SLbA and the EIE-LVC kit.

a<p>Parameters was calculated using all samples presented in this work for TL (CT + CD + CL + ML. n = 135). VL (CT + CD + VL. n = 125) and CVL (CT + CD + CVL. n = 75).</p><p>*<i>Cut-off</i> obtained by ROC curve.</p>#<p><i>Cut off</i> obtained according to the manufacturer.</p><p>Abbreviations: Tse; total sensitivity; TSp: total specificity; CI: confidence interval; PPV: positive predictive value; NPV: negative predictive value; AC: accuracy.</p><p>Diagnostic performance of <i>r</i>CatL, peptide-1, SLbA and the EIE-LVC kit.</p

Comparison of the ELISA reactivity of <i>r</i>CatL, Peptide-1, SLbA and the EIE-LVC kit against sera from TL and VL patients and from <i>L. infantum</i>-infected dogs.

<p><b>TL and VL</b>: ELISAs were performed on samples from different groups of individuals (CT, control group; CD, Chagas disease patients; CL, cutaneous leishmaniasis; ML, mucosal leishmaniasis; VL, visceral leishmaniasis). <b>CVL</b>: ELISAs were performed on samples from different groups of dogs (CT, control group; CD, <i>T. cruzi</i>-infected dogs; CVL, canine visceral leishmaniasis). ^*Cut-off obtained by a ROC curve. ^#Cut-off suggested by the manufacturer.</p

Primers and sequence information.

<p>Primers and sequence information.</p

Evaluation of RNAi-mediated impairment of Pgp 4 and 12 expression in the WT and IVR30 strain.

<p>(A) Percentage of L₃ paralysis percentage after 48 hours ivermectin exposure for groups Pgps 4 and 12 silenced [RNAi⁺], Pgps 4 and 12 non silenced [RNAi^-] and negative control [CT], cultured only in M9 medium and not exposed to ivermectin treatment. (B) Assessement of L₃ viability cell using propidium iodide marker after 48 hours ivermectin exposure in Pgps 4 and 12 silenced [RNAi⁺] and Pgps 4 and 12 non silenced [RNAi^-] groups. P value refers to one-way ANOVA test with Turkey’s multiple comparisons test.</p

<i>In silico</i> analysis revealing a close relationship between Pgps from <i>C</i>. <i>elegans</i> and nematodes parasites represented.

<p>As follow: <i>Ascaris suum</i> (A), <i>Brugia malayi</i> (B), <i>Cooperia oncophora</i> (C), <i>Haemonchus contortus</i> (H), <i>Loa loa</i> (L), <i>Onchocerca volvulus</i> (O), <i>Parascaris equorum</i> (P), <i>Strongyloides ratti</i> (S), <i>Trichuris trichiura</i> (T), <i>Wuchereria bancrofti</i> (W), <i>C</i>. <i>brenneri</i> (1), <i>C</i>. <i>briggsae</i> (2), <i>C</i>. <i>remanei</i> (4), Pgp 4 of <i>C</i>. <i>elegans</i> (Δ), Pgp 12 of <i>C</i>. <i>elegans</i> (•), all remaining Pgps from <i>C</i>. <i>elegans</i> (2).</p

<i>C</i>. <i>elegans</i> Pgps genes profile using 2^-ΔΔCt method.

<p>Pgps gene expression for WT strains are represented by dashed line. P values were obtained with One sample t test, comparing sample value to WT expression reference value.</p