Incidence of Leishmania donovani infection and Visceral Leishmaniasis (VL) was assessed in a prospective study in Indian and Nepalese high-endemic villages. DAT-seroconversion was used as marker of incident infection in 3 yearly surveys. The study population was followed up to month 30 to identify incident clinical cases. In a cohort of 9034 DAT-negative individuals with neither active signs nor history of VL at baseline, 42 VL cases and 375 asymptomatic seroconversions were recorded in the first year, giving an infection∶disease ratio of 8.9 to 1. In the 18 months' follow-up, 7 extra cases of VL were observed in the seroconverters group (N = 375), against 14 VL cases among the individuals who had not seroconverted in the first year (N = 8570) (RR = 11.5(4.5<RR<28.3)). Incident asymptomatic L. donovani infection in VL high-endemic foci in India and Nepal is nine times more frequent than incident VL disease. About 1 in 50 of these new but latent infections led to VL within the next 18 months. Visceral Leishmaniasis is well known as a public health problem in North-Indian Bihar state and adjacent districts in Nepal, with about 300.000 new cases per year. As not all infections with L.donovani lead to disease, the impact of control programs should not only be measured in numbers of VL cases, but also in number of new infections. So far there have been little or no data on the infection∶disease ratio for this region, and the evolution of these latent infections. Using DAT seroconversion as a marker of infection, we found incident asymptomatic infection to be nine times more frequent than incident VL disease in high-endemic villages in India and Nepal, and about 1 in 50 of these latent infections lead to VL in the next 18 months, while over 80% turn seronegative again within a year. Asymptomatic DAT-positivity detected through screening in a person with no history of VL, and especially in case of documented recent seroconversion, is a risk factor for ultimately developing VL. Further studies on transient and persistent asymptomatic L. donovani infection are needed to better understand their immunological patterns and serokinetics, their level of infectivity, and their potential for later progression to VL.

Objective To test the effectiveness of large scale distribution of longlasting nets treated with insecticide in reducing the incidence of visceral leishmaniasis in India and Nepal. Design Paired cluster randomised controlled trial designed to detect a 50% reduction in incidence of Leishmania donovani infection. Setting Villages in Muzaffarpur district in India and Saptari, Sunsari, and Morang districts in Nepal. Participants 13 intervention and 13 control clusters. 12 691 people were included in the analysis of the main outcome (infection), and 19 810 were enrolled for the secondary (disease) end point. Intervention Longlasting insecticidal nets (treated with deltamethrin) were distributed in the intervention clusters in December 2006. Main outcome measures Infection was determined by direct agglutination test at 12 and 24 months after the intervention in those who had negative results (titre <1:1600) at baseline. The effect estimate was computed as the geometric mean of the risk ratios for seroconversion for each cluster pair (net/no net), with its 95% confidence interval. Formal tests of effect of no intervention were obtained with a paired t test. Results There was no significant difference in the risk of seroconversion over 24 months in intervention (5.4%; 347/6372) compared with control (5.5%; 345/6319 people) clusters (risk ratio 0.90, 95% confidence interval 0.49 to 1.65) nor in the risk of clinical visceral leishmaniasis (0.99, 0.46 to 1.40). Adjustment for covariates did not alter these conclusions. Conclusions There is no evidence that large scale distribution of longlasting insecticidal nets provides additional protection against visceral leishmaniasis compared with existing control practice in the Indian subcontinent. The observed effect was small and not significant, though the confidence intervals did not exclude a 50% change in either direction. Trial registration Clinical Trials NCT 2005-015374.

Depressed cell-mediated immunity in human visceral leishmaniasis (VL) (also known as kala-azar), revealed as the inability of peripheral blood mononuclear cells (PBMCs) to respond toLeishmaniaantigen, remains a hallmark of and is thought to underlie the progressive nature of this disease. We recently reported the ability of a whole-blood, gamma interferon (IFN-γ) release assay to detect subclinical infections among healthy individuals living in an area where kala-azar is endemic (Bihar, India) and the surprising result that patients with active VL also secreted significant levels of antigen-specific IFN-γ in this assay. We were interested in ascertaining whether these findings would be true for a larger cohort of subjects and in employing the whole-blood assay to detect additional cytokines that might better correlate with the disease status of infected individuals. We evaluated IFN-γ, tumor necrosis factor alpha (TNF-α), and interleukin-10 (IL-10) release in 35 patients with active VL, 54 patients with VL who were cured, 27 patients with other diseases, 52 healthy controls who lived in regions where VL or kala-azar is not endemic (NEHCs [for nonendemic healthy controls]), and 147 healthy controls who lived in regions where kala-azar is endemic (EHCs [for endemic healthy controls]). The cellular responses of the EHCs were correlated with their serological antibody titers againstLeishmania donovaniandPhlebotomus argentipessaliva. The whole-blood cells from the majority of both active (80%) and cured (85%) VL patients, as well as 24% of EHCs with presumed subclinical infections, produced significantly elevated levels of IFN-γ. The findings do not support a severe Th1 response defect in kala-azar. Importantly, only the patients with active VL also produced IL-10, which in conjunction with IFN-γ better reflects the immune responses that distinguish individuals with active disease from cured or subclinically infected, immune individuals.

Visceral leishmaniasis (VL) is associated with increased circulating levels of multiple pro-inflammatory cytokines and chemokines, including IL-12, IFNγ, and TNFα, and elevated expression of IFNγ mRNA in lesional tissue such as the spleen and bone marrow. However, an immunological feature of VL patients is that their peripheral blood mononuclear cells (PBMCs) typically fail to respond to stimulation with leishmanial antigen. Unexpectedly, it was recently shown that Leishmania specific IFNγ, can readily be detected when a whole blood stimulation assay (WBA) is used. We sought to define the conditions that permit whole blood cells to respond to antigen stimulation, and clarify the biological role of the IFNγ found to be released by cells from VL patients. CD4+ T cells were found to be crucial for and the main source of the IFNγ production in Leishmania stimulated whole blood (WB) cultures. Complement, antibodies and red blood cells present in whole blood do not play a significant role in the IFNγ response. The IFNγ production was reduced by blockade of human leukocyte antigen (HLA)-DR, indicating that the response to leishmanial antigens observed in WB of active VL patients is a classical HLA- T cell receptor (TCR) driven reaction. Most importantly, blockade of IFNγ in ex-vivo splenic aspirate cultures demonstrated that despite the progressive nature of their disease, the endogenous IFNγ produced in patients with active VL serves to limit parasite growth. Our research aims to understand the immune failure underlying progression of human visceral leishmaniasis (VL). A key immunological feature of VL patients is that their peripheral blood mononuclear cells (PBMCs) do not respond to stimulation with leishmanial antigen. Surprisingly, when employing a whole blood assay we discovered significant levels of IFNγ in response to soluble Leishmania donovani antigen (WBA) in VL patients. We were interested to understand the relevance of the IFNγ to the anti-parasitic response. Animal models and in vitro studies have shown that IFNγ is a key effector cytokine required for control of the infection, however, the role of endogenous IFNγ in control of parasites in VL patients, has not been demonstrated. Our results show that CD4 cells were required for and were the source of Leishmania specific IFNγ in WBA of VL patients. Optimal IFNγ response required interaction with HLA-DR, supporting that VL is not due to an intrinsic Th1 response defect per se. The Leishmania driven IFNγ appears to limit parasite growth in patients with active VL, since blockade of IFNγ ex-vivo in splenic aspirate cultures enhanced parasite survival. This suggests that IFNγ may have been prematurely dismissed as an adjunct therapy in treatment of VL.

Antibody (IgG) responses to the saliva of Phlebotomus argentipes were investigated using serum samples from regions of India endemic and non-endemic for visceral leishmaniasis (VL). By pre-adsorbing the sera against the saliva of the competing human-biting but non-VL vector P. papatasi, we significantly improved the specificity of a P. argentipes saliva enzyme-linked immunosorbent assay. Using this method, we observed a statistically significant correlation between antibodies to P. argenitpes saliva and the average indoor density of female sand flies. Additionally, the method was able to detect recent changes in vector exposure when sera from VL patients were assayed before, during, and after hospitalization and protected from sand fly bites under untreated bed nets. Collectively, these results highlight the utility of antibodies to P. argentipes saliva as an important tool to evaluate VL vector control programs.

Visceral leishmaniasis is the world' second largest vector-borne parasitic killer and a neglected tropical disease, prevalent in poor communities. Long-lasting insecticidal nets (LNs) are a low cost proven vector intervention method for malaria control; however, their effectiveness against visceral leishmaniasis (VL) is unknown. This study quantified the effect of LNs on exposure to the sand fly vector of VL in India and Nepal during a two year community intervention trial. As part of a paired-cluster randomized controlled clinical trial in VL-endemic regions of India and Nepal we tested the effect of LNs on sand fly biting by measuring the antibody response of subjects to the saliva of Leishmania donovani vector Phlebotomus argentipes and the sympatric (non-vector) Phlebotomus papatasi. Fifteen to 20 individuals above 15 years of age from 26 VL endemic clusters were asked to provide a blood sample at baseline, 12 and 24 months post-intervention. A total of 305 individuals were included in the study, 68 participants provided two blood samples and 237 gave three samples. A random effect linear regression model showed that cluster-wide distribution of LNs reduced exposure to P. argentipes by 12% at 12 months (effect 0.88; 95% CI 0.83–0.94) and 9% at 24 months (effect 0.91; 95% CI 0.80–1.02) in the intervention group compared to control adjusting for baseline values and pair. Similar results were obtained for P. papatasi. This trial provides evidence that LNs have a limited effect on sand fly exposure in VL endemic communities in India and Nepal and supports the use of sand fly saliva antibodies as a marker to evaluate vector control interventions. Visceral leishmaniasis (VL), also known as kala azar, is one of the major public health concerns of the Indian subcontinent, caused by Leishmania donovani transmitted by the bite of the sand fly Phlebotomus argentipes. To date, Indoor Residual Spraying (IRS) campaigns have been unable to control the disease. This makes Long-lasting insecticidal nets (LNs) an attractive alternative or complement to IRS. Therefore, it is important to assess the extent that LNs reduce bites from P. argentipes. When female sand flies bite they require their saliva to efficiently bloodfeed. For humans and animals alike, the host' immune response against components of sand fly saliva can be used as a marker of exposure to the vector. Here we describe how comprehensive coverage of LNs in trial communities over two years reduced antibody levels to the saliva of P. argentipes and P. papatasi (a man-biting sand fly that co-exists with P. argentipes but does not transmit VL) sand flies by 9–12% compared to communities without LNs. Our results demonstrate that the large-scale distribution of LNs did not confer significant additional protection against sand fly bites in VL-endemic regions of India and Nepal and questions the indoor transmission of L. donovani in these regions.