General characteristics of human T-cell lymphotropic virus type 1 (HTLV-1)-infected and -uninfected groups (n = 122).

<p>General characteristics of human T-cell lymphotropic virus type 1 (HTLV-1)-infected and -uninfected groups (n = 122).</p

Vestibular-evoked myogenic potential triggered by galvanic vestibular stimulation (galvanic-VEMP) tracings.

<p>(A) Delayed short-latency (SL) and medium-latency (ML) waves in an individual with asymptomatic human T-cell lymphotropic virus type 1 (HTLV-1) infection. (B) Absent SL response with delayed ML response latency in an individual with possible HTLV-1-associated myelopathy (HAM). (C) Absent SL and ML responses in an individual with HAM. These recordings were obtained from subjects with their heads rotated to the left and electromyographic (EMG) responses recorded from the right gastrocnemius muscle. The black line indicates the trace recorded with the cathode and anode on the right and left side respectively, whereas the gray line indicates the opposite stimulation polarity. SL, short-latency response onset; ML, medium-latency response onset.</p

Comparative analysis of the latency and amplitude of short-latency (SL) and medium-latency (ML) responses between human T-cell lymphotropic virus type 1 (HTLV-1)-infected and -uninfected groups.

<p>Comparative analysis of the latency and amplitude of short-latency (SL) and medium-latency (ML) responses between human T-cell lymphotropic virus type 1 (HTLV-1)-infected and -uninfected groups.</p

Frequency comparisons between normal and altered vestibular-evoked myogenic potential triggered by galvanic vestibular stimulation (galvanic-VEMP) for each group.

<p>The type of response (normal, delayed latency, or absent) and its frequency (%) is shown for each group. G1, uninfected control group; G2, human T-cell lymphotropic virus type 1 (HTLV-1)-asymptomatic group; G3, possible HTLV-1-associated myelopathy (HAM) group; G4, HAM group; n, number of participants. ^aDelayed latency of short-latency (SL), medium-latency (ML), or both responses. ^bAbsent SL, ML, or both responses. *p<0.001, chi-square or Fisher’s exact test.</p

Flow cytometry-based triplex array (FC-TRIPLEX) for selective analyses of <i>L</i>. <i>infantum</i> promastigotes, <i>T</i>. <i>cruzi</i> epimastigotes, and <i>L</i>. <i>braziliensis</i> promastigotes.

<p>(A) Representative dot plots demonstrating similarities in the morphometric features of the fixed Trypanosomatidae organisms. (B) Differential fluorescence staining with fluorescein isothiocyanate (FITC) (<i>L</i>. <i>infantum</i> = 0.0 μg/mL, <i>T</i>. <i>cruzi</i> = 2.0 μg/mL, or <i>L</i>. <i>braziliensis</i> = 20.0 μg/mL) or Alexa Fluor 647 (<i>L</i>. <i>infantum</i> = 0.0 μg/mL, <i>T</i>. <i>cruzi</i> = 0.78 μg/mL, or <i>L</i>. <i>braziliensis</i> = 12.5 μg/mL) as a strategy for segregating parasites based on the FL1 and FL4 fluorometric profiles, respectively. (C) Unidimensional histograms for the FL1 and FL4 profiles illustrating the stability of fluorescently stained single or premixed parasite suspensions during storage at room temperature (RT), 4°C, or −20°C.</p

Experimental design for simultaneous detection of anti-Trypanosomatidae IgG1 antibodies by flow cytometry-based triplex array (FC-TRIPLEX Chagas/Leish IgG1).

<p>(A) Major assay steps. PBS, phosphate-buffered saline; FBS, fetal bovine serum; PE, phycoerythrin. (B) Parasite gating strategy based on FCS versus SSC dot plot distribution followed by a set marker setup for internal control positivity limit, leading to a Percentage of Positive Fluorescent Parasites value of <2.0%. (C) This set marker setup was maintained to subsequently determine disease-related reactivity patterns. VL, visceral leishmaniasis; CH, Chagas disease; LCL, localized cutaneous leishmaniasis; NI, noninfected controls. Dark frames highlight species-specific IgG1 reactivity patterns at selected serum dilutions (VL, anti-<i>L</i>. <i>infantum</i> at 1:32,000; CH, anti-<i>T</i>. <i>cruzi</i> at 1:2,000; LCL and NI, anti-<i>L</i>. <i>braziliensis</i> at 1:1,000) according to Garcia et al. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122938#pone.0122938.ref013" target="_blank">13</a>], Matos et al. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122938#pone.0122938.ref015" target="_blank">15</a>], and Pereira et al. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122938#pone.0122938.ref012" target="_blank">12</a>], respectively.</p

Flow cytometry-based inverted detuned algorithm for analysis of anti-Trypanosomatidae IgG1 reactivity for differential diagnosis of Chagas disease and leishmaniasis.

<p>(A) Reactivity board showing the pathway for achieving distinctive disease-related positive results (triangles) and negative results (circles) results and avoiding inconclusive reactivity (squares). VL, visceral leishmaniasis; CH, Chagas disease; LCL, localized cutaneous leishmaniasis; NI, noninfected controls. (B) Proposed inverted detuned algorithm starting at the selection of the <i>L</i>. <i>infantum</i> population on FL2 versus FL4 bidimensional distribution followed by analyses of IgG1 reactivity at 1:32,000 on unidimensional FL2 histograms expressed as percentage of fluorescence positive parasites (PPFP). A PPFP value of >60% defines the VL diagnosis, whereas a PPFP value of ≤60% leads to the next algorithm step. Selection of the <i>T</i>. <i>cruzi</i> population is followed by analyses of IgG1 reactivity at 1:2,000, with a PPFP value of >50% defining the CH diagnosis, and a PPFP value of ≤50% leading to the next algorithm stage. At the final step <i>L</i>. <i>braziliensis</i> population selection is followed by analyses of IgG1 reactivity at 1:1,000, with a PPFP value of >60% defining the LCL diagnosis, and a PPFP value of ≤60% excluding all three Trypanosomatidae infections. (C) Global accuracy analyses underscored the outstanding overall performance of the method, which reached 95% (correct results were obtained for 76 of the 80 serum samples).</p

Species-specific anti-Trypanosomatidae IgG1 reactivity and flow cytometry-based triplex array (FC-TRIPLEX Chagas/Leish IgG1) performance indexes.

<p>(A) Titration curve averages of percentages of positive fluorescent parasites (PPFP) for anti-Trypanosomatidae IgG1 in serum samples from patients with visceral leishmaniasis (VL, black circles), patients with Chagas disease (CH, dark gray circles), localized cutaneous leishmaniasis (LCL, light gray circles), and noninfected controls (NI, white circles). (B) Scattering plots for individuals at selected sera dilutions highlighted by gray round rectangles (VL, anti-<i>L</i>. <i>infantum</i> at 1:32,000; CH, anti-<i>T</i>. <i>cruzi</i> at 1:2,000; LCL and NI, anti-<i>L</i>. <i>braziliensis</i> at 1:1,000) using the cut-off edges (dotted lines), previously described by Garcia et al. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122938#pone.0122938.ref013" target="_blank">13</a>], Matos et al. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122938#pone.0122938.ref015" target="_blank">15</a>], and Pereira et al. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122938#pone.0122938.ref012" target="_blank">12</a>]. PPFP = 60% for anti-<i>L</i>. <i>infantum</i> IgG1; PPFP = 50% for anti-<i>T</i>. <i>cruzi</i> IgG1; and PPFP = 60% for anti-<i>L</i>. <i>braziliensis</i>, respectively. (C) Receiver operating characteristic, ROC curve analyses confirmed the previously selected cut-off and demonstrated outstanding performance indexes: Se, sensitivity; Sp, specificity; PPV, positive predictive value; NPV, negative predictive value; AUC, area under the curve; LR+ and LR–, positive and negative likelihood ratios, respectively, for anti-<i>L</i>. <i>infantum</i> and anti-<i>T</i>. <i>cruzi</i> IgG1.</p