Irreversibility of treatment with 17-AAG on intracellular <i>Leishmania</i>.

<p>To assessment the reversibility of parasite growth inhibition by treatment with 17-AAG parasite load was determined by quantifying the percentage of infected macrophages (<b>A</b>) and the number of parasites per macrophage (<b>B</b>) as described in Materials and Methods. Bars represent means ± SD of one representative experiment out of two performed in sextuplicate (one-way ANOVA, ***<i>p</i><0.0001, Dunnett’s Multiple Comparison Test, *<i>p</i><0.05, **<i>p</i><0.001, ***<i>p</i><0.0001, post-test for linear trend, <i>p</i><0.0001).</p

Alterations suggestive of autophagy in intracellular parasites treated with 17-AAG.

<p>Transmission electron microscopy was used to investigate ultrastructural morphological alterations in intracellular parasites inside 17-AAG-treated macrophages. (<b>A</b>) Control infected macrophages. After 12 h of treatment, several morphological alterations were seen in intracellular parasites, including: (<b>B</b>) numerous small vesicles some containing cytoplasmic material inside <b>(black arrow)</b>, (<b>C</b>) vacuoles larger in size <b>(black arrow-head)</b>, (<b>D</b>) with intravacuolar materials degraded <b>(white arrow)</b>. After 24 h of treatment, the intracellular parasites presented a large number of vesicles occupying most of the cytoplasm containing well-preserved nuclei, mitochondria, and subpellicular microtubules (<b>E–F</b>). After 48 h of treatment, no preserved parasites inside cells were observed, yet empty vesicles, and membrane-bounded structures with an electron-density similar to parasite cytosol in parasitophorous vacuoles were seen (<b>G</b>). At 12 and 24 h after treatment, several alterations were also visible in parasite cytoplasm, including myelin figures <b>(*)</b> (<b>H–I</b>), vesicles with double-layered membranes <b>(white arrow-head)</b> (<b>J–M</b>)<b>,</b> and portions of mitochondria inside membrane-bounded structures (<b>M</b>). The nuclei (<b>N</b>) and mitochondria (<b>M</b>) and kinetoplast (<b>K</b>) remained intact in all groups.</p

Reduction of parasite intracellular viability by 17-AAG.

<p>Treatment’s effect on parasite viability was assessed after 24 h (<b>A</b>) and 48 h (<b>B</b>) of infection, as described in Materials and Methods. Bars represent means ± SD of one representative experiment out of two performed in sextuplicate (one-way ANOVA, ***<i>p</i><0.0001, Dunnett’s Multiple Comparison Test, *<i>p</i><0.05, **<i>p</i><0.001, ***<i>p</i><0.0001, post-test for linear trend, <i>p</i><0.0001; Mann Whitney test, **<i>p</i><0.001).</p

Modulation of mediator production by treatment with 17-AAG.

<p>Mediators released by 17-AAG-treated cells were measured in cell supernatants using an inflammatory CBAKit, as described in Materials and Methods: (<b>A</b>) IL-6; (<b>B</b>) IL-10; (<b>C</b>) IL-12; (<b>D</b>) TNF-α; (<b>E</b>) MCP-1. Bars represent means ± SD of a single experiment performed in sextuplicate (Student’s <i>t</i>-test and Mann-Whitney, **<i>p</i><0.001, ***<i>p</i><0.0001).</p

Inhibition of axenic growth of <i>Leishmania</i> and reduction in parasite load by 17-AAG.

<p>(<b>A</b>) Axenic promastigotes were exposed to several concentrations of 17-AAG (25, 125, 500 nM) for 48 h and the number of viable parasites was assessed as described in Materials and Methods. Data are presented as the percentage inhibition of parasite growth related to untreated controls (4,754×10⁷). Bars represent means ± SD of one representative out of two experiments performed in sextuplicate (one-way ANOVA, Dunnett’s Multiple Comparison Test, ***<i>p</i><0.0001, post-test for linear trend, <i>p</i><0.0001). (<b>B, C</b>) Drug effects at early times after infection. Following 6 h of incubation with parasites, macrophage cultures were treated for 6, 24 and 48 h with specific concentrations of 17-AAG (25, 125, 500 nM); (<b>D, E</b>) Drug effects at later stages after infection. Following 6 h of incubation with parasites, macrophage cultures were reincubated for additional 48 h then submitted to treatment with specific concentrations of 17-AAG (25, 125, 500 nM). Bars represent means ± SD of one representative experiment out of two performed in sextuplicate (one-way ANOVA, Dunnett’s Multiple Comparison Test, *<i>p</i><0.05, **<i>p</i><0.001, ***<i>p</i><0.0001, post-test for linear trend, <i>p</i><0.0001).</p

Reduced O₂

<p>⁻<b>and NO production in macrophage cultures treated with 17-AAG.</b> (<b>A</b>) O₂⁻ production was measured at early stages of infection using a lucigenin-derived CL method. Points on the graph represent photon emissions per second by macrophage cultures 2 min prior to the addition of <i>L. amazonensis</i> promastigotes, as well as throughout the incubation period of 20 min. Data are derived from one representative experiment out of four performed in uniplicate (Mann Whitney test, p = 0.028); (<b>B</b>) Intracellular O₂⁻ production was assessed by determining cell fluorescence in the presence of hydroethidine (5 µM) and expressed as MFI using a flow cytometer. The histogram overlay depicts the MFI of hydroethidine-labeled cells (solid lines) in comparison to unlabeled control cells (shaded areas). Data are derived from one representative experiment out of three performed in uniplicate (Mann Whitney test, <i>p</i> = 1). (<b>C</b>) NO production was measured by detecting nitrite in the supernatants of 17-AAG-treated cells, as described in Materials and Methods. Bars represent NO production measurement expressed as means ± SD of one representative experiment out of four performed in triplicate or more (Student’s <i>t</i> test, ***<i>p</i><0.0001).</p

The Rapid Test Based on <i>Leishmania infantum</i> Chimeric rK28 Protein Improves the Diagnosis of Canine Visceral Leishmaniasis by Reducing the Detection of False-Positive Dogs

<div><p>Visceral Leishmaniasis (VL) has spread to many urban centers worldwide. Dogs are considered the main reservoir of VL, because canine cases often precede the occurrence of human cases. Detection and euthanasia of serologically positive dogs is one of the primary VL control measures utilized in some countries, including Brazil. Using accurate diagnostic tests can minimize one undesirable consequence of this measure, culling false-positive dogs, and reduce the maintenance of false-negative dogs in endemic areas. In December 2011, the Brazilian Ministry of Health replaced the ELISA (EIE CVL) screening method and Indirect Immunofluorescence Test (IFI CVL) confirmatory method with a new protocol using the rapid DPP CVL screening test and EIE CVL confirmatory test. A study of diagnostic accuracy of these two protocols was done by comparing their performance using serum samples collected from a random sample of 780 dogs in an endemic area of VL. All samples were evaluated by culture and real time PCR; 766 out of the 780 dogs were tested using the previous protocol (IFI CVL + EIE CVL) and all 780 were tested using the current protocol (DPP CVL + EIE CVL). Performances of both diagnostic protocols were evaluated using a latent class variable as the gold standard. The current protocol had a higher specificity (0.98 vs. 0.95) and PPV (0.83 vs. 0.70) than the previous protocol, although sensitivity of these two protocols was similar (0.73). When tested using sera from asymptomatic animals, the current protocol had a much higher PPV (0.63 vs. 0.40) than the previous protocol (although the sensitivity of either protocol was the same, 0.71). Considering a range of theoretical CVL prevalences, the projected PPVs were higher for the current protocol than for the previous protocol for each theoretical prevalence value. The findings presented herein show that the current protocol performed better than previous protocol primarily by reducing false-positive results.</p></div

Percent of positive results of five CVL diagnostic tests performed on canine sera samples from an endemic area of Camaçari.

<p>Percent of positive results of five CVL diagnostic tests performed on canine sera samples from an endemic area of Camaçari.</p

The mass use of deltamethrin collars to control and prevent canine visceral leishmaniasis: A field effectiveness study in a highly endemic area

<div><p>Background</p><p>Visceral leishmaniasis (VL) is a zoonosis of great importance. Limitations in current VL control measures compromise efficacy, indicating the need to implement new strategies. The aim of this study was to evaluate the effectiveness of the mass use of deltamethrin-impregnated collars in dogs as a public health measure to control and prevent canine visceral leishmaniasis (CVL).</p><p>Methodology</p><p>An interventional study was implemented in two endemic areas in the district of Monte Gordo (Bahia-Brazil): an intervention area, in which VL seronegative dogs were collared, and a control area in which only conventional CVL control measures were applied. At baseline, seropositive dogs were removed and seronegative dogs were included. Dogs were then reevaluated every 7–8 months for almost two years. At each time point, dogs in the intervention area that remained seronegative received new collars and newly identified seronegative dogs were included and collared. The local zoonosis control authorities were notified of any dogs that tested seropositive in both areas, which were subsequently marked for euthanasia as mandated by the Brazilian Ministry of Health.</p><p>Principal findings</p><p>In the first serological survey, seroprevalence was similar in both areas. At the second evaluation, significant reductions in seroprevalence were seen in both areas, while seroprevalence in the intervention area reduced to 6.0% during the final evaluation versus an increase of 11.0% in the control area. This significant increase and the estimated relative risk (RR = 0.55) indicated protection against CVL in the intervention area. Although CVL incidence did not differ significantly between the areas, an increased tendency was observed in the control area, which could be due to low seroconversion rates throughout the study or a high loss to follow-up.</p><p>Conclusions/Significance</p><p>Although our evaluation of the effectiveness of deltamethrin-impregnated collars as a community-wide public health control measure was inconclusive, this measure likely provides protection over time. In endemic areas of Brazil, this strategy represents an operational challenge for local zoonosis control authorities, indicating the need for adjustments, including improved collar design.</p></div

The mass use of deltamethrin collars to control and prevent canine visceral leishmaniasis: A field effectiveness study in a highly endemic area - Fig 2

<p>Identification of the intervention area (A) and control area (B) in the district of Monte Gordo–municipality of Camaçari (Bahia-Brazil). Source: USGS LandsatLook (<a href="https://landsatlook.usgs.gov/viewer.html" target="_blank">https://landsatlook.usgs.gov/viewer.html</a>).</p