Search

Background: Nepal launched a visceral leishmaniasis (also known as kala-azar) elimination initiative in 2005. We primarily aimed to assess whether transmission of Leishmania donovani had decreased since the launch of the initiative. We also assessed the validity of the direct agglutination test (DAT) as a marker of infection, in view of future surveillance systems., Methods: We did a repeat survey in a population aged 2 years and older for whom baseline serological data were available from 2006. Data were from three districts in the eastern region of Nepal. The primary outcome of interest was prevalent infection with L donovani as measured with DAT (cutoff value ≥1:3200). We compared age group-specific and cluster-specific seroprevalences in 2016 with those in 2006, using χ 2 tests, with a specific focus on the comparison of seroprevalences in children born between 1996 and 2005, and those born between 2006 and 2015. To estimate the overall adjusted risk ratio for being seropositive in 2016 compared with 2006, we fitted a Poisson model controlling for age, sex, and cluster., Findings: Between Oct 17, 2016, and Dec 26, 2016, we assessed 6609 individuals. DAT prevalence in children younger than 10 years was 4·1% (95% CI 3·2-5·4) in 2006 versus 0·5% (0·1-1·7) in 2016 (p<0·0001). Seroprevalence was lower in 2016 than in 2006 in all age groups and in all repeated clusters. The overall adjusted risk ratio of being seropositive was 0·44 (95% CI 0·37-0·52) for 2016 compared with 2006, and 0·04 (0·01-0·16) in children younger than 10 years., Interpretation: Our findings show that transmission of L donovani in Nepal has decreased significantly between 2006 and 2016, coinciding with the elimination programme. DAT seems useful for monitoring of L donovani transmission., Funding: The Directorate-General for Development Cooperation of Belgium., (Copyright © 2020 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY-NC-ND 4.0 license. Published by Elsevier Ltd.. All rights reserved.)

Background: Asymptomatic Leishmania donovani infections outnumber clinical presentations, however the predictors for development of active disease are not well known. We aimed to identify serological, immunological and genetic markers for progression from L. donovani infection to clinical Visceral Leishmaniasis (VL)., Methods: We enrolled all residents >2 years of age in 27 VL endemic villages in Bihar (India). Blood samples collected on filter paper on two occasions 6-12 months apart, were tested for antibodies against L. donovani with rK39-ELISA and DAT. Sero converters, (negative for both tests in the first round but positive on either of the two during the second round) and controls (negative on both tests on both occasions) were followed for three years. At the start of follow-up venous blood was collected for the following tests: DAT, rK39- ELISA, Quantiferon assay, SNP/HLA genotyping and L.donovani specific quantitative PCR., Results: Among 1,606 subjects enrolled,17 (8/476 seroconverters and 9/1,130 controls) developed VL (OR 3.1; 95% CI 1.1-8.3). High DAT and rK39 ELISA antibody titers as well as positive qPCR were strongly and significantly associated with progression from seroconversion to VL with odds ratios of 19.1, 30.3 and 20.9 respectively. Most VL cases arose early (median 5 months) during follow-up., Conclusion: We confirmed the strong association between high DAT and/or rK39 titers and progression to disease among asymptomatic subjects and identified qPCR as an additional predictor. Low predictive values do not warrant prophylactic treatment but as most progressed to VL early during follow-up, careful oberservation of these subjects for at least 6 months is indicated., Competing Interests: The authors have declared that no competing interests exist.

Leishmania donovani causes visceral leishmaniasis (VL), the second most deadly vector-borne parasitic disease. A recent epidemic in the Indian subcontinent (ISC) caused up to 80% of global VL and over 30,000 deaths per year. Resistance against antimonial drugs has probably been a contributing factor in the persistence of this epidemic. Here we use whole genome sequences from 204 clinical isolates to track the evolution and epidemiology of L. donovani from the ISC. We identify independent radiations that have emerged since a bottleneck coincident with 1960s DDT spraying campaigns. A genetically distinct population frequently resistant to antimonials has a two base-pair insertion in the aquaglyceroporin gene LdAQP1 that prevents the transport of trivalent antimonials. We find evidence of genetic exchange between ISC populations, and show that the mutation in LdAQP1 has spread by recombination. Our results reveal the complexity of L. donovani evolution in the ISC in response to drug treatment.

Background: In the Indian subcontinent, Visceral leishmaniasis is endemic in a geographical area coinciding with the Lower Gangetic Plain, at low altitude. VL occurring in residents of hill districts is therefore often considered the result of Leishmania donovani infection during travel. Early 2014 we conducted an outbreak investigation in Okhaldhunga and Bhojpur districts in the Nepal hills where increasing number of VL cases have been reported., Methodology/principal Findings: A house-to-house survey in six villages documented retrospectively 35 cases of Visceral Leishmaniasis (VL). Anti-Leishmania antibodies were found in 22/23 past-VL cases, in 40/416 (9.6%) persons without VL and in 12/155 (7.7%) domestic animals. An age- and sex- matched case-control study showed that exposure to known VL-endemic regions was no risk factor for VL, but having a VL case in the neighbourhood was. SSU-rDNA PCR for Leishmania sp. was positive in 24 (5%) of the human, in 18 (12%) of the animal samples and in 16 (14%) bloodfed female Phlebotomus argentipes sand flies. L. donovani was confirmed in two asymptomatic individuals and in one sand fly through hsp70-based sequencing., Conclusions/significance: This is epidemiological and entomological evidence for ongoing local transmission of L. donovani in villages at an altitude above 600 meters in Nepal, in districts considered hitherto non-endemic for VL. The VL Elimination Initiative in Nepal should therefore consider extending its surveillance and control activities in order to assure VL elimination, and the risk map for VL should be redesigned.

There is increasing interest in the role of asymptomatic infection in transmission of Visceral Leishmaniasis (VL). We studied the individual, household and environmental factors associated with asymptomatic Leishmania donovani infected individuals and VL. 7,538 individuals living in VL endemic villages in India and Nepal were divided into three mutually exclusive groups based on their VL history and Direct Agglutination Test (DAT) results in yearly serosurveys over a two-year period. The groups were (1) VL cases, (2) asymptomatically infected individuals (seroconverters) and (3) seronegative individuals. VL cases and seroconverters were compared to seronegative individuals in mixed logistic regression models. The risk of seroconversion and disease was significantly increased in individuals aged 14 to 24 years old and by the presence of other DAT-positive, asymptomatically infected individuals and VL cases in the house. The risk of seroconversion was higher in Indian than in Nepalese villages and it increased significantly with age, but not so for VL. This study demonstrates that, when risk factors for leishmanial infection and VL disease are evaluated in the same population, epidemiological determinants for asymptomatic infection and VL are largely similar.

Introduction: Asymptomatic persons infected with the parasites causing visceral leishmaniasis (VL) usually outnumber clinically apparent cases by a ratio of 4-10 to 1. We assessed the risk of progression from infection to disease as a function of DAT and rK39 serological titers., Methods: We used available data on four cohorts from villages in India and Nepal that are highly endemic for Leishmania donovani. In each cohort two serosurveys had been conducted. Based on results of initial surveys, subjects were classified as seronegative, moderately seropositive or strongly seropositive using both DAT and rK39. Based on the combination of first and second survey results we identified seroconvertors for both markers. Seroconvertors were subdivided in high and low titer convertors. Subjects were followed up for at least one year following the second survey. Incident VL cases were recorded and verified., Results: We assessed a total of 32,529 enrolled subjects, for a total follow-up time of 72,169 person years. Altogether 235 incident VL cases were documented. The probability of progression to disease was strongly associated with initial serostatus and with seroconversion; this was particularly the case for those with high titers and most prominently among seroconvertors. For high titer DAT convertors the hazard ratio reached as high as 97.4 when compared to non-convertors. The strengths of the associations varied between cohorts and between markers but similar trends were observed between the four cohorts and the two markers., Discussion: There is a strongly increased risk of progressing to disease among DAT and/or rK39 seropositives with high titers. The options for prophylactic treatment for this group merit further investigation, as it could be of clinical benefit if it prevents progression to disease. Prophylactic treatment might also have a public health benefit if it can be corroborated that these asymptomatically infected individuals are infectious for sand flies.

Unlabelled: Leishmania donovani is an intracellular protozoan parasite that causes leishmaniasis, which can range from a self-healing cutaneous disease to a fatal visceral disease depending on the infecting species. Miltefosine is currently the latest and only oral antileishmanial that came out of drug discovery pipelines in the past few decades, but recent reports indicate a significant decline in its efficacy against visceral leishmaniasis (also known as kala-azar) in the Indian subcontinent. This relapse rate of up to 20% within 12 months after treatment was shown not to be related to reinfection, drug quality, drug exposure, or drug-resistant parasites. We therefore aimed to assess other phenotypes of the parasite that may affect treatment outcome and found a significant association between the number of metacyclic parasites, parasite infectivity, and patient treatment outcome in the Indian subcontinent. Together with previous studies on resistance of L. donovani against pentavalent antimonials, these data suggest that the infectivity of the parasite, or related phenotypes, might be a more determinant factor for treatment failure in visceral leishmaniasis than drug susceptibility, warranting a reassessment of our current view on treatment failure and drug resistance in leishmaniasis and beyond., Importance: The high miltefosine relapse rate poses a major challenge for the current Kala-Azar Elimination Program in the Indian subcontinent and other leishmaniasis control programs worldwide. This relapse rate could not be related to reinfection, drug-resistant parasites, or reduced treatment quality. Here we report that an increased infectivity of the parasite is associated with miltefosine relapse of visceral leishmaniasis (VL) patients. These results supplement those obtained with antimonial-resistant L. donovani where an increased infectivity was also observed. This challenges the current view of Leishmania drug susceptibility being the biggest parasitic factor that contributes to treatment failure in leishmaniasis. These selected more infectious parasites may pose an additional burden to leishmaniasis control programs, highlighting the importance of multifaceted control measures to achieve leishmaniasis elimination in the Indian subcontinent and other regions where leishmaniasis is endemic.

Objectives: Recent epidemiological reports indicate that asymptomatic human infections with Leishmania donovani, the causative agent of visceral leishmaniasis or Kala-azar (KA), occur frequently in India. We explored markers of infection., Methods: Blood samples were collected from 286 healthy subjects from 16 villages in the Muzaffarpur district of Bihar. These individuals were classified into three groups: (i) persons with no history of KA and living in a house where no KA cases were previously reported, (ii) persons with no history of KA but living in a house where KA cases were diagnosed at the time of sampling or in the past, and (iii) successfully treated KA patients. Each sample was tested using a Leishmania-specific PCR to detect Leishmania DNA, and two serological tests to demonstrate anti-Leishmania antibodies: the Direct Agglutination Test and rK39 ELISA., Results: PCR positivity was similar among the three groups (20-25%). In contrast, among treated patients, the percentage of serologically positive individuals was roughly five times that of healthy individuals with no KA history, as measured with either test. Living in a house where KA had been reported did not affect seropositivity., Conclusion: A significant proportion of asymptomatic infections of Leishmania exist in endemic regions. Using a combination of molecular and serological tests increases the capacity to detect infections at different stages. Further work is required to understand the kinetics of the markers., (© 2013 Blackwell Publishing Ltd.)

We assessed the prevalence of post-kala-azar dermal leishmaniasis (PKDL), a late cutaneous manifestation of visceral leishmaniasis (VL), in 16 VL-endemic communities in Bihar, India. The prevalence of confirmed PKDL cases was 4.4 per 10 000 individuals and 7.8 if probable cases were also considered. The clinical history and treatment of the post-kala-azar dermal leishmaniasis cases are discussed., (© 2012 Blackwell Publishing Ltd.)

The current standard to assess pentavalent antimonial (SSG) susceptibility of Leishmania is a laborious in vitro assay of which the result has little clinical value because SSG-resistant parasites are also found in SSG-cured patients. Candidate genetic markers for clinically relevant SSG-resistant parasites identified by full genome sequencing were here validated on a larger set of clinical strains. We show that 3 genomic locations suffice to specifically detect the SSG-resistant parasites found only in patients experiencing SSG treatment failure. This finding allows the development of rapid assays to monitor the emergence and spread of clinically relevant SSG-resistant Leishmania parasites.

Recent clinical isolates of Leishmania donovani from the hyperendemic zone of Bihar were characterised in vitro in terms of their sensitivity towards sodium stibogluconate in a macrophage culture system. The resulting half maximal effective concentration (EC(50)) values were compared with those of known sensitive isolates. Fifteen of the isolates showed decreased sensitivity towards SSG with an average EC(50) of 25.7 ± 4.5 μg/ml pentavalent antimony (defined as antimony resistant), whereas nine showed considerable sensitivity with an average EC(50) of 4.6 ± 1.7 μg/ml (defined as antimony sensitive). Out of those nine, seven were recent clinical isolates and the remaining two were known sensitive isolates. Compared with the antimony sensitive, resistant isolates showed enhanced expression of thiol metabolising enzymes in varying degrees coupled with increased intracellular non-protein thiol content, decreased fluorescence anisotropy (inversely proportional with membrane fluidity) and over-expression of the terminal glycoconjugates (N-acetyl-d-galactosaminyl residue). Macrophages infected with resistant but not with sensitive showed up-regulation of the ATP Binding Cassette transporter multidrug resistance protein 1 and permeability glycoprotein, while the supernatant contained abundant IL-10. The above results reinforce the notion that antimony resistant parasites have undergone a number of biochemical and biophysical changes as part of their adaptation to ensure their survival in the host., (Copyright © 2011 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.)

Background: In the Indian subcontinent, about 200 million people are at risk of developing visceral leishmaniasis (VL). In 2005, the governments of India, Nepal and Bangladesh started the first regional VL elimination program with the aim to reduce the annual incidence to less than 1 per 10,000 by 2015. A mathematical model was developed to support this elimination program with basic quantifications of transmission, disease and intervention parameters. This model was used to predict the effects of different intervention strategies., Methods and Findings: Parameters on the natural history of Leishmania infection were estimated based on a literature review and expert opinion or drawn from a community intervention trial (the KALANET project). The transmission dynamic of Leishmania donovani is rather slow, mainly due to its long incubation period and the potentially long persistence of parasites in infected humans. Cellular immunity as measured by the Leishmanin skin test (LST) lasts on average for roughly one year, and re-infection occurs in intervals of about two years, with variation not specified. The model suggests that transmission of L. donovani is predominantly maintained by asymptomatically infected hosts. Only patients with symptomatic disease were eligible for treatment; thus, in contrast to vector control, the treatment of cases had almost no effect on the overall intensity of transmission., Conclusions: Treatment of Kala-azar is necessary on the level of the individual patient but may have little effect on transmission of parasites. In contrast, vector control or exposure prophylaxis has the potential to efficiently reduce transmission of parasites. Based on these findings, control of VL should pay more attention to vector-related interventions. Cases of PKDL may appear after years and may initiate a new outbreak of disease; interventions should therefore be long enough, combined with an active case detection and include effective treatment.

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

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.

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.

Visceral leishmaniasis (VL) is an important vector-borne disease caused by Leishmania donovani in the Indian subcontinent. The actual incidence and role of asymptomatic infections in the region are not wellknown. We used the direct agglutination test (DAT) and the rK39 ELISA as L. donovani infection markers in 10 VL endemic villages in Nepal. DAT titre distribution showed two subgroups in the population (infected and non-infected individuals), while rK39 did not. The agreement between both tests was moderate (j = 0.53; 95% CI 0.49–0.57). More research is needed to develop validated markers for Leishmania infection.

Summary: Visceral leishmaniasis (VL) is a major public health problem in the Indian subcontinent where the Leishmania donovani transmission cycle is described as anthroponotic. However, the role of animals (in particular domestic animals) in the persistence and expansion of VL is still a matter of debate. We combined Direct Agglutination Test (DAT) results in humans and domestic animals with Geographic Information System technology (i.e. extraction maps and scan statistic) to evaluate the exposure to L. donovani on these 2 populations in a recent VL focus in Nepal. A Poisson regression model was used to assess the risk of infection in humans associated with, among other factors, the proportion of DAT-positive animals in the proximities of the household. The serological results showed that both humans and domestic animals were exposed to L. donovani. DAT-positive animals and humans were spatially clustered. The presence of serologically positive goats (IRR=9.71), past VL cases (IRR=2.62) and the proximity to a forest island dividing the study area (IRR=3.67) increased the risk of being DAT-positive in humans. Even if they are not a reservoir, domestic animals, and specially goats, may play a role in the distribution of L. donovani, in particular in this new VL focus.

Objective: Visceral Leishmaniasis (VL) is highly prevalent in Bihar, India. India and its neighbours aim at eliminating VL, but several knowledge gaps in the epidemiology of VL may hamper that effort. The prevalence of asymptomatic infections with Leishmania donovani and their role in transmission dynamics are not well understood. We report data from a sero-survey in Bihar., Methods: Demographic and immunological surveys were carried out in July and November 2006, respectively in 16 highly VL endemic foci in Muzaffarpur district in Bihar. Household and individual information was gathered and capillary blood samples were collected on filter papers. Direct agglutination test (DAT) was used to determine infected individuals (cut-off titre 1:1600). DAT results were tabulated against individual and household variables. A multivariate generalized estimating equation (GEE) model was used to study the prevalence of serologically positive individuals taking into account the clustering at household and cluster levels., Results: Of study subjects 18% were DAT positive, and this proportion increased with age. Women had a significantly lower prevalence than men >14 years old. Owning domestic animals (cows, buffaloes or goats) was associated with a higher risk of being DAT positive [OR 1.16 (95% CI 1.01-1.32)], but socio-economic status was not., Conclusions: Prevalence of leishmanial antibodies was high in these communities, but variable. Demographic factors (i.e. marriage) may explain the lower DAT positivity in women >14 years of age. Within these homogeneously poor communities, socio-economic status was not linked to L. donovani infection risk at the individual level, but ownership of domestic animals was.

Objective: Nepal reports a visceral leishmaniasis (VL) incidence of 5 per 10 000 per year on the basis of notification by health facilities, but little community-based epidemiological information exists. We report data on prevalence rates of Leishmania donovani infection in ten communities in East Nepal., Methods: Ten clusters with highest VL incidence rates were purposefully selected in Nepal. All households were mapped and socio-demographic data and data on past VL incidence were collected. An exhaustive serological survey was performed of individuals aged >2 years, by collecting finger prick blood on filter paper in November-December 2006. The samples were tested by direct agglutination, and a titre >or=1:1600 was taken as marker of infection. A generalized estimating equation (GEE) model was used to assess risk factors for Direct Agglutination Test (DAT) positivity taking into account the clustering at household and village level., Results: The sero-survey (n = 5397) showed an infection prevalence rate of 9%, (range 5-15% per cluster) with higher prevalence in men (9.9%) than in women (8.3%) (P = 0.049). Male gender, increasing age and poverty were significant risk factors in the final GEE model., Conclusion: Leishmania infection rate in high-transmission areas in Nepal is associated with gender, age and socio-economic status.