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

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.

ABSTRACTDepressed 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.

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.

IntroductionAsymptomatic persons infected with the parasites causing visceral leishmaniasis (VL) usually outnumber clinically apparent cases by a ratio of 4–10 to 1. We describe patterns of markers of Leishmania donovani infection and clinical VL in relation to age in Bihar, India.MethodsWe selected eleven villages highly endemic for Leishmania donovani. During a 1-year interval we conducted two house to house surveys during which we collected blood samples on filter paper from all consenting individuals aged 2 years and above. Samples were tested for anti-leishmania serology by Direct Agglutination Test (DAT) and rK39 ELISA. Data collected during the surveys included information on episodes of clinical VL among study participants.ResultsWe enrolled 13,163 persons; 6.2% were reactive to DAT and 5.9% to rK39. Agreement between the tests was weak (kappa = 0.30). Among those who were negative on both tests at baseline, 3.6% had converted to sero-positive on either of the two tests one year later. Proportions of sero-positives and sero-converters increased steadily with age. Clinical VL occurred mainly among children and young adults (median age 19 years).DiscussionAlthough infection with L. donovani is assumed to be permanent, serological markers revert to negative. Most VL cases occur at younger ages, yet we observed a steady increase with age in the frequency of sero-positivity and sero-conversion. Our findings can be explained by a boosting effect upon repeated exposure to the parasite or by intermittent release of parasites in infected subjects from safe target cells. A certain proportion of sero-negative subjects could have been infected but below the threshold of antibody abundance for our serologic testing.

In this phase III trial for diagnostics for visceral leishmaniasis (VL) in India, we compared parasitological diagnosis with several serological tests: direct agglutination test (freeze dried; DAT-FD), rK-39 strip test, rK-26 strip test and a latex agglutination test for antigen detection in urine (KAtex) in 452 subjects from the endemic regions of Bihar, India. The subjects were segregated into four categories: 230 confirmed patients, 52 probable cases, 70 non-cases and 100 healthy endemic controls. The first two groups were used for estimating sensitivity, the latter two for specificity. Sensitivity of DAT-FD was 98.9%, rK-39: 98.9%, KAtex: 67.0% and rK-26: 21.3%. Sensitivity of DAT-FD on blood taken on filter paper (DAT-FDF) was 99.3%, which was comparable with that using serum. Specificity of serological tests was comparable and high (DAT-FD and DAT-FDF: 94%, rK-39 strip test: 97%, KAtex: 99% and rK-26 strip test: 100%). The classical 'gold standard' parasitological demonstration in splenic smear performed poorly as it missed 18.4% of cases that benefited from VL treatment. Reproducibility of the serological tests between field and central laboratories was excellent (kappa = 1.0, 0.99, 0.96 and 0.94 respectively for microscopy, DAT-FD, rK-39 strip test and rK-26 strip test). A high degree of agreement was observed between DAT-FD and rK-39 strip test (kappa = 0.986). Although DAT-FD and rK-39 strip test were highly sensitive with excellent specificity, the ease of use of the latter makes it most suitable for the diagnosis of VL in the field conditions.

The persistence of anti-Leishmania donovani antibodies in past visceral leishmaniasis (VL) cases was retrospectively assessed by means of the direct agglutination test (DAT) and the rK39 enzyme-linked immunosorbent assay (ELISA). Antibody titers remained high for an extended period of time in past cases of VL. These results highlight the need to carefully elicit the history of patients with VL symptoms.