Pushkar Shivam

DDT-based indoor residual spraying suboptimal for visceral leishmaniasis elimination in India

Significance Visceral leishmaniasis (VL), also known as kala-azar, is a major parasitic disease on the Indian subcontinent, with 85% of the disease incidence in India. As with malaria, indoor residual spraying (IRS) can be used to control and, ultimately, eliminate VL as a public health problem. However, in India, widespread resistance to dichlorodiphenyltrichloroethane (DDT), the insecticide used, combined with poor quality assurance (QA) of IRS and limited entomological surveillance, is hindering the VL elimination effort. Here, we present entomological abundance and insecticide resistance data and data arising from QA of IRS to explore these issues and establish an evidence base for improving the Indian VL elimination program. Unless these issues are addressed, the South Asian VL elimination effort will fail. Indoor residual spraying (IRS) is used to control visceral leishmaniasis (VL) in India, but it is poorly quality assured. Quality assurance was performed in eight VL endemic districts in Bihar State, India, in 2014. Residual dichlorodiphenyltrichloroethane (DDT) was sampled from walls using Bostik tape discs, and DDT concentrations [grams of active ingredient per square meter (g ai/m2)] were determined using HPLC. Pre-IRS surveys were performed in three districts, and post-IRS surveys were performed in eight districts. A 20% threshold above and below the target spray of 1.0 g ai/m2 was defined as “in range.” The entomological assessments were made in four districts in IRS and non-IRS villages. Vector densities were measured: pre-IRS and 1 and 3 mo post-IRS. Insecticide susceptibility to 4% DDT and 0.05% deltamethrin WHO-impregnated papers was determined with wild-caught sand flies. The majority (329 of 360, 91.3%) of pre-IRS samples had residual DDT concentrations of <0.1 g ai/m2. The mean residual concentration of DDT post-IRS was 0.37 g ai/m2; 84.9% of walls were undersprayed, 7.4% were sprayed in range, and 7.6% were oversprayed. The abundance of sand flies in IRS and non-IRS villages was significantly different at 1 mo post-IRS only. Sand flies were highly resistant to DDT but susceptible to deltamethrin. The Stockholm Convention, ratified by India in 2006, calls for the complete phasing out of DDT as soon as practical, with limited use in the interim where no viable IRS alternatives exist. Given the poor quality of the DDT-based IRS, ready availability of pyrethroids, and susceptibility profile of Indian sand flies, the continued use of DDT in this IRS program is questionable.

Leishmania donovani skews the CD56+ Natural Killer T cell response during human visceral leishmaniasis

The objective of this study was to understand the categorical function of CD4(+)CD56(+) and CD8(+)CD56(+) NKT cells in human visceral leishmaniasis. These cell populations were significantly deregulated in human peripheral blood during VL. The in vitro experiments showed that CD4(+)NKT cells, but not CD8(+)NKT cells, migrated towards the Leishmania donovani infection site. Additionally, CD4(+)NKT cells from VL subjects primarily expressed CD25(+)Foxp3 and IL-10 compared with healthy subjects. However, CD8(+)NKT cells expressed primarily IFN-γ and killer cell immunoglobulin receptor compared with healthy subjects. Because the ratio of CD4(+) and CD8(+)NKT cells was 1:10, adoptive transfer of CD3(+)CD56(+) NKT cells effectively reduced the L. donovani burden in infected macrophages. This study concludes that although CD4(+)NKT cells are pathogenic and accumulate at the infection site, CD8(+)NKT cells may be protective in contact with target cells.

Development of a Simple Dipstick Assay for Operational Monitoring of DDT

Background Indoor residual spraying (IRS) of DDT is used to control visceral leishmaniasis (VL) in India. However, the quality of spraying is severely compromised by a lack of affordable field assays to monitor target doses of insecticide. Our aim was to develop a simple DDT insecticide quantification kit (IQK) for monitoring DDT levels in an operational setting. Methodology/ principle findings DDT quantification was based on the stoichiometric release of chloride from DDT by alkaline hydrolysis and detection of the released ion using Quantab chloride detection strips. The assay was specific for insecticidal p,p`-DDT (LoQ = 0.082 g/m2). Bostik discs were effective in post spray wall sampling, extracting 25–70% of active ingredient depending on surface. Residual DDT was sampled from walls in Bihar state in India using Bostik adhesive discs and DDT concentrations (g p,p`-DDT/m2) were determined using IQK and HPLC (n = 1964 field samples). Analysis of 161 Bostik samples (pooled sample pairs) by IQK and HPLC produced excellent correlation (R2 = 0.96; Bland-Altman bias = −0.0038). IQK analysis of the remaining field samples matched HPLC data in identifying households that had been under sprayed, in range or over sprayed. Interpretation A simple dipstick assay has been developed for monitoring DDT spraying that gives comparable results to HPLC. By making laboratory-based analysis of DDT dosing accessible to field operatives, routine monitoring of DDT levels can be promoted in low- and middle- income countries to maximise the effectiveness of IRS.

Identification of B-cell Epitope of Leishmania donovani and its application in diagnosis of visceral leishmaniasis

Diagnosis of visceral leishmaniasis (VL) is often hindered by cross-reactions with antigens from other related parasite infections. This study aimed to develop an immunochromatographic test (ICT) which can detect the antigen present in circulating immune complexes (CICs) of VL patients using B-cell epitope-specific antibodies. MS analysis of six immunoreactive 2DE spots revealed two epitopes i.e. RFFVQGDGIGQHSLQEALERR (P1) and RRVAVLVLLDRL (P2) (From a hypothetical protein [Acc No: XP_003861458.1]). The epitope conservancy analysis suggested that the linear epitope (P1P2) is 97–100% conserved among Leishmania species and diverged from Homo sapiens (61% query coverage and 80% identity). Further, immunoinformatics analysis of hydrophilicity and flexibility confirmed the antigenicity of the peptide fragment. The linear epitope (P1P2) was synthesized (98% purity) and the purity was confirmed by high-performance liquid chromatography and MS. The indirect Enzyme linked immunosorbent assay results confirmed the presence of the corresponding antibody in VL patient’s sera but not in those of healthy and other diseases. The result demonstrated a sensitivity 90%; Se Cl95% (82.16–96.27)% and specificity 100%; Sp Cl95% (84.56–100)% which indicated the possibility to be used as a diagnostic tool. Sensitivity, specificity, and diagnostic efficiency of colloidal gold conjugated anti-P1P2 antibody ICT strip was 100, 95.2, and 96.7%, respectively, which is slightly better as compared to other ICT for VL. Though, our result indicated the utility of anti-P1P2 antibody to detect CICs epitopes, a large-scale inspection in endemic and non-endemic area and in different ethnic population is needed for its validation and authentication.

Leishmania donovani: Influence of anti-leishmanial therapy on expression of lymphocyte function-associated antigen-3 and its relevance to pathogenisis in visceral leishmaniasis

Lymphocyte function associated antigen 3 (LFA-3) is known as adhesion molecule with its role in T-cell activation signaling as well as in Foxp3 expression. Its influences on IL-10 production is also available, whose role in pathogenesis is well documented. However, this molecule is not yet directly addressed for its association with visceral leishmaniasis (VL). We investigated the relationship between Leishmania donovani infection and expression of LFA-3 in VL patients in their pre and post treatment stage through this case control study. Present study reports L. donovani mediated expression of LFA-3 on CD14(+) monocytes in human VL. Active cases of VL was observed with 2.91-fold increased mean florescence intensity (MFI) of LFA-3 expression on CD14(+) cells compared to healthy control (p = 0.0001). This increased MFI of untreated VL cases was reduced 1.92-fold in successfully treated cases (p = 0.0001). This observation was also accorded by mRNA expression for LFA-3 in monocytes of corresponding samples. The expression of LFA-3 was also observed influenced by L. donovani load in splenic aspirates, as it was 1.71-fold elevated in patients with Ld grade ≥3+ compared to patients with ≤2+ Ld grade (p = 0.0121). To evaluate the possibility that L. donovani utilize LFA-3 mediated evasion pathway in human visceral leishmaniasis; in vitro experiments were performed for measurement of pathogenic cytokine IL-10 and Foxp3 mRNA expression. The IL-10 production and Foxp3 expression in peripheral blood mononuclear cells from VL subjects were observed regulated significantly (p = 0.0131 and 0.0436 when compared with untreated samples) in presence of an antagonist to LFA-3. This study recommends further investigations to strengthen the pathogenic and prognostic significance of LFA-3 in visceral leishmaniasis.

Leishmania donovani resistant to Ambisome or Miltefosine exacerbates CD58 expression on NK cells and promotes trans-membrane migration in association with CD2

HighlightsHigher expression of CD58 but not of CD2 was observed on CD56+ cells during Visceral Leishmaniasis.Expression of CD58 on CD56+ cells were further exacerbated in Ambisome or Miltefosine relapsed VL.Ratio of CD56+CD58+IFN‐&ggr;+/CD56+CD58+IL‐10+ cells reduces after stimulation with Ld.Ambisome or Miltefosine resistance Ld significantly increase CD58 expression on CD56+ cells.Antagonist to CD58 or CD2, down‐regulates CD56+ cell recruitment at Ld infection site. Abstract Visceral leishmaniasis (VL) is a disease that is associated with compromised immunity and drug un‐responsiveness as well as with the emergence of drug resistance in Leishmania donovani (Ld). Ld down‐modulates cellular immunity by manipulating signaling agents, including a higher expression of the adhesion molecule CD58. The expression of CD58 and CD2 on natural killer (NK) cells facilitates intercellular adhesion and signaling. The influence of drug‐resistant Ld on the expression of CD58 and CD2 was addressed in this study. The mean florescence intensity (MFI) of CD58 but not of CD2 was twofold higher on CD56+ cells during VL, but was down‐regulated after treatment. In addition, MFI of CD58 on CD56+ cells was further exacerbated in VL subjects who had relapsed after Ambisome or Miltefosine treatment. The same pattern of CD58 expression was also obtained upon stimulation of healthy peripheral blood mononuclear cells with Miltefosine‐ or Ambisome‐resistant Ld. The ratio of CD56+CD58+IFN‐&ggr;+/CD56+CD58+IL‐10+ cells was reduced by 6.98‐fold after stimulation with Ld. Further, an antagonist to CD58 or its counter‐receptor CD2 down‐regulated CD56+ NK cell recruitment across a polycarbonate trans‐membrane at Ld infection sites. This study reports that factors associated with drug resistance in Ld probably promote higher expression of CD58 on CD56+ cells and their migration to the infection site in association with CD2.

CD8 dim but not CD8 bright cells positive to CD56 dominantly express KIR and are cytotoxic during visceral leishmaniasis

This study reports a structural and functional heterogeneity of CD8+CD56+NKT cells, which usually decrease quantitatively during visceral leishmaniasis. Based on fluorescence intensity of CD8 receptors on CD56+NKT cells, two populations of CD8+CD56+NKT cells have been identified. These cells were recognized as CD8dimCD56+NKT and CD8brightCD56+NKT cells. We further analyzed the functional nature of CD8dim and CD8bright positive CD56+NKT cells. In comparison to CD8brightCD56+NKT cells, a significantly higher percentage of CD8dimCD56+NKT cells expressed KIR during VL. The percentage of CD8dimCD56+NKT cells expressing KIR was found 4 fold higher in VL as compared to healthy subjects. But, the difference was insignificant in case of CD8brightCD56+NKT cells. CD8+CD56+NKT cells release granzyme B to kill the infected cells. A categorical difference was also observed in the function of CD8dimCD56+NKT and CD8brightCD56+NKT cells during visceral leishmaniasis. The percentage of granzyme B expressing CD8dimCD56+NKT cells was 2.83 fold higher in VL compared to healthy subjects. But, there was no significant difference in granzyme B expressing CD8brightCD56+NKT cells in samples from healthy and VL subjects. However, within VL subject, the percentage of granzyme B expressing CD8dimCD56+NKT cells was 5.7 fold higher in comparison to CD8brightCD56+NKT cells. This study concludes that CD8dimCD56+NKT cells are more cytotoxic than CD8brightCD56+NKT cells during VL.

Visceral leishmaniasis: A novel nuclear envelope protein ‘nucleoporins-93 (NUP-93)’ from Leishmania donovani prompts macrophage signaling for T-cell activation towards host protective immune response

&NA; The shift of macrophage and T‐cell repertoires towards proinflammatory cytokine signalling ensures the generation of host‐protective machinery that is otherwise compromised in cases of the intracellular Leishmania parasite. Different groups have attempted to restore host protective immunity. These vaccine candidates showed good responses and protective effects in murine models, but they generally failed during human trials. In the present study, we evaluated the effect of 97 kDa recombinant nucleoporin‐93 of Leishmania donovani (rLd‐NUP93) on mononuclear cells in healthy and treated visceral leishmaniasis (VL) patients and on THP‐1 cell lines. rLd‐NUP93 stimulation increased the expression of the early lymphocyte activation marker CD69 on CD4+ and CD8+ T cells. The expression of the host protective pro‐inflammatory cytokines IFN‐&ggr;, IL‐12 and TNF‐&agr; was increased, with a corresponding down‐regulation of IL‐10 and TGF‐&bgr; upon rLd‐NUP93 stimulation. This immune polarization resulted in the up‐regulation of NF‐&kgr;B p50 with scant expression of SMAD‐4. Augmenting lymphocyte proliferation upon priming with rLd‐NUP93 ensured its potential for activation and generation of strong T‐cell mediated immune responses. This stimulation extended the leishmanicidal activity of macrophages by releasing high amounts of reactive oxygen species (ROS). Further, the leishmanicidal activity of macrophages was intensified by the elevated production of nitric oxide (NO). The fact that this antigen was earlier reported in circulating immune complexes of VL patients highlights its antigenic importance. In addition, in silico analysis suggested the presence of MHC class I and II‐restricted epitopes that proficiently trigger CD8+ and CD4+ T‐cells, respectively. This study reported that rLd‐NUP93 was an effective immunoprophylactic agent that can be explored in future vaccine design.

Leishmania donovani mediated higher expression of CCL4 induces differential accumulation of CD4+CD56+NKT and CD8+CD56+NKT cells at infection site

&NA; Sterile cure from visceralized Leishmania donovani (L. donovani) needs Th1 cell support along with the assistance from innate immune cells, NK cells and NKT cells. NKT cells play as a connecting link between innate and adaptive immune cell and support T helper cell function. Earlier, a categorical function of CD56 positive CD4+ or CD8+ NKT cells was reported in visceral leishmaniasis (VL). It was observed in in vitro that CD4+CD56+NKT cells, but not CD8+CD56+NKT cells, were accumulated at the L. donovani infection site. Therefore, in vitro experiments have been carried out to decipher the mechanism behind preferential accumulation of CD4+CD56+NKT cells at infection site. In this study, 1.89 fold higher expression of CCL4/MIP‐1&bgr; was noticed in infected macrophages. The higher expression of CCL4 was correlated with preferential accumulation of CCR5+CD4+CD56+NKT cells and apoptosis of CD8+CD56+NKT cells at in vitro infection site. The CD4+CD56+NKT cells were also observed expressing TGF‐&bgr; dominantly. Interaction of CCL4 chemotaxis was interrupted by blocking, which led to drift back the TGF‐&bgr; producing CD4+CD56+NKT cells and promoted CD8+CD56+NKT cells recruitment in in vitro infection site. CCR5 blockade also reduced CD25 and FoxP3 positive CD4+CD56+NKT cells in in vitro infection site. Therefore, it was concluded that Leishmania promotes strategic expression of CCL4, which alternately attracts CCR5+ cells, mostly expressing regulatory cytokines, at infection site. This reduces the CD8+CD56+NKT cells at infection site through Smad4 mediated TGF‐&bgr; expression and activation of caspases. Data indicates that L. donovani induces higher expression of CCL4 in host cell to attract CCR5+ cells under its strategic plan to downregulate host immune response.