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

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

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

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

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

Sequences of primers used for quantification of mRNA expression by real-time PCR^a.

a<p>F: Forward primer, R: Reverse primer. GenBank accession number of the sequence used to design primers and their product length are shown, as well as each PCR efficiency and <i>R²</i>.</p

Histopathological and parasite density analyses of skin of dogs naturally infected with <i>L. infantum</i>.

<p>Animals were categorized according to their clinical status into asymptomatic (AD, <i>n</i> = 10), oligosymptomatic (OD, <i>n</i> = 10), or symptomatic (SD, <i>n</i> = 15) or categorized according to skin parasite density into low (LP, <i>n</i> = 12), medium (MP, <i>n</i> = 11), or high (HP, <i>n</i> = 12) parasite density. The control group is represented by CD (<i>n</i> = 16). Photomicrographs of cutaneous cellular infiltrates from dogs naturally inflected by <i>L. infantum</i> stained by hematoxylin and eosin (<b>A</b>). Representative cellular infiltrates of study groups are depicted: Control dogs (1 and 2 ); AD or LP (3 and 4); OD or MP (5 and 6); SD or HP (7 and 8). <b>Left panels:</b> Slides shown at 10× magnification; bar, 100 µm. <b>Right panels:</b> Slides shown at 40× magnification; bar, 25 µm. Correlation between quantitative analysis of cutaneous cellular infiltrate with clinical status (<b>B</b>) or skin parasite density (<b>C</b>) are presented. Correlation between clinical groups and skin parasite density is also presented (<b>D</b>). The results are expressed as the mean number of cells present in cutaneous cellular infiltrates evaluated at 20 fields plus standard deviation. In (<b>D</b>), the results are expressed as the mean of the log number of skin parasite density plus standard deviation. Significant differences (<i>p</i><0.05) compared with CD and AD or LP groups are indicated by symbols * and #, respectively. Spearman's correlation indexes (<i>r</i> and <i>p</i> values) are shown on the graphs when applicable.</p

Chemokines in <i>Leishmania spp</i>. infection.

<p>Chemokines in <i>Leishmania spp</i>. infection.</p

Correlation between parasite density (LDU) and cell-types presenting into skin from <i>L. infantum</i> infected dogs.

<p>The results were expressed on graphs as scattering of individual values. Spearman correlation indexes (<i>r</i>) at <i>p</i><0.05 are shown on graphs. Connecting lines illustrated positive and negative correlation indexes.</p