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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.

Objective: To assess the rate of default from treatment in the visceral leishmaniasis (VL) elimination programme and to identify risk factors and its underlying causes., Methods: Case-control study conducted between December 2009 and June 2012 in three primary health centres (PHCs) of Muzaffarpur district, India. Patients who defaulted from treatment from the PHCs were considered as 'cases' and those who completed their treatment as 'controls'. Two controls were included in the study for each case. Respondents' opinion and satisfaction with the services provided at the PHCs were also elicited. Logistic regression was performed to assess the contribution of sociodemographic variables on patient status, and a discriminant analysis was used (after decomposing the original data) to identify factors that can predict the patient status as defaulter or not, based on factor scores of the components as predictor variables., Results: During the study period, 16.3% (89/544) of patients defaulted; 87 cases and 188 controls were interviewed through a semistructured questionnaire. Women tended to be more at risk for default (OR, 1.6, 95% CI (0.9 -2.9). Treatment received was miltefosine in 55.6% and sodium stibogluconate (SSG) in 44.4%. Most (86%) defaulters completed their treatment at other healthcare facilities; 70% of them preferred non-governmental institutions. Most cited reasons for default were seeking a second opinion for VL treatment and preferring to be treated in specialised VL centres. Discriminant analysis showed only one significant predictor: dissatisfaction with the medical care received in PHCs., Conclusion: Efforts are needed to enhance the quality of VL care at PHC level, which will be beneficial in increasing treatment completion rates., (© 2017 John Wiley & Sons Ltd.)

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.

The Muzaffarpur-TMRC Health and Demographic Surveillance System (HDSS), established in 2007, was developed as an enlargement of the scope of a research collaboration on the project Visceral Leishmaniasis in Bihar, which had been ongoing since 2005. The HDSS is located in a visceral leishmaniasis (VL)-endemic area in the Muzaffarpur district of Bihar state in India. It is the only HDSS conducting research on VL, which is a vector-borne infectious disease transmitted by female phlebotomine sandflies and is fatal if left untreated. Currently the HDSS serves a population of over 105,000 in 66 villages. The HDSS collects data on vital events including pregnancies, births, deaths, migration and marriages, as well as other socio-economic indicators, at regular intervals. Incident VL cases are identified. The HDSS team is experienced in conducting both qualitative and quantitative studies, sample collection and rapid diagnostic tests in the field. In each village, volunteers connect the HDSS team with the community members. The Muzaffarpur-TMRC HDSS provides opportunities for studies on VL and other neglected tropical diseases (NTDs) and their interaction with demographic events such as migration. Queries related to research collaborations and data sharing can be sent to Dr Shyam Sundar at [drshyamsundar@hotmail.com]., (© The Author 2014; all rights reserved. Published by Oxford University Press on behalf of the International Epidemiological Association.)

Background: Recent reports indicated high miltefosine treatment failure rates for visceral leishmaniasis (VL) on the Indian subcontinent. To further explore the pharmacological factors associated with these treatment failures, a population pharmacokinetic-pharmacodynamic study was performed to examine the relationship between miltefosine drug exposure and treatment failure in a cohort of Nepalese patients with VL., Methods: Miltefosine steady-state blood concentrations at the end of treatment were analyzed using liquid chromatography tandem mass spectrometry. A population pharmacokinetic-pharmacodynamic analysis was performed using nonlinear mixed-effects modeling and a logistic regression model. Individual estimates of miltefosine exposure were explored for their relationship with treatment failure., Results: The overall probability of treatment failure was 21%. The time that the blood concentration was >10 times the half maximal effective concentration of miltefosine (median, 30.2 days) was significantly associated with treatment failure: each 1-day decrease in miltefosine exposure was associated with a 1.08-fold (95% confidence interval, 1.01-1.17) increased odds of treatment failure., Conclusions: Achieving a sufficient exposure to miltefosine is a significant and critical factor for VL treatment success, suggesting an urgent need to evaluate the recently proposed optimal allometric miltefosine dosing regimen. This study establishes the first evidence for a drug exposure-effect relationship for miltefosine in the treatment of VL.

Background: High frequency of relapse in miltefosine-treated visceral leishmaniasis (VL) patients in India and Nepal followed up for twelve months., Objective: To identify epidemiological and clinical risk factors for relapse of VL in patients recently treated with standard dosing of miltefosine in India and Nepal., Design: Prospective observational study in three Primary Health Centers and one reference center in Muzaffarpur district, Bihar, India; and two zonal hospitals and a university hospital in South-east Nepal; records of all consenting patients diagnosed with VL and treated with miltefosine according to the current treatment guidelines of the Kala azar elimination program between 2009 and 2011., Results: We compared the clinical records of 78 cases of relapse with those of 775 patients who had no record of subsequent relapse. Relapse was 2 times more common amongst male patients (IRR 2.14, 95% CI 1.27-3.61), and 2 to 3 times more frequent in the age groups below 15 compared to the over 25 year olds (age 10 to 14: IRR 2.53; 95% CI 1.37-4.65 and Age 2 to 9: IRR 3.19; 95% CI 1.77-5.77). History of earlier VL episodes, or specific clinical features at time of diagnosis such as duration of symptoms or spleen size were no predictors of relapse., Conclusions: Young age and male gender were associated with increased risk of VL relapse after miltefosine, suggesting that the mechanism of relapse is mainly host-related i.e. immunological factors and/or drug exposure (pharmacokinetics). The observed decrease in efficacy of miltefosine may be explained by the inclusion of younger patients compared to the earlier clinical trials, rather than by a decreased susceptibility of the parasite to miltefosine. Our findings highlight the importance of proper clinical trials in children, including pharmacokinetics, to determine the safety, efficacy, drug exposure and therapeutic response of new drugs in this age group.

The elimination of infectious diseases requires reducing transmission below a certain threshold. The Visceral Leishmaniasis (VL) Elimination Initiative in Southeast Asia aims to reduce the annual VL incidence rate below 1 case per 10,000 inhabitants in endemic areas by 2015 via a combination of case management and vector control. Using a previously developed VL transmission model, we investigated transmission thresholds dependent on measures reducing the sand fly density either by killing sand flies (e.g., indoor residual spraying and long-lasting insecticidal nets) or by destroying breeding sites (e.g., environmental management). Model simulations suggest that elimination of VL is possible if the sand fly density can be reduced by 67% through killing sand flies, or if the number of breeding sites can be reduced by more than 79% through measures of environmental management. These results were compared to data from two recent cluster randomised controlled trials conducted in India, Nepal and Bangladesh showing a 72% reduction in sand fly density after indoor residual spraying, a 44% and 25% reduction through the use of long-lasting insecticidal nets and a 42% reduction after environmental management. Based on model predictions, we identified the parameters within the transmission cycle of VL that predominantly determine the prospects of intervention success. We suggest further research to refine model-based predictions into the elimination of VL.

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.

Promastigote miltefosine (MIL) susceptibility was performed on Leishmania donovani isolates from Indian patients with visceral leishmaniasis treated with MIL. Isolates that were obtained before the onset of MIL treatment, after completion of treatment (29th day), or at the time of treatment failure, were screened using in vitro promastigote assay. The MIL susceptibility of the pre-treatment isolates (N = 24, mean IC50 ± SEM = 3.74 ± 0.38 μM) was significantly higher than that of the post-treatment group (N = 26, mean IC50 ± SEM = 6.15 ± 0.52 μM; P = 0.0006) but was similar in the cured patients (N = 22, mean IC50 ± SEM = 5.58 ± 0.56 μM) and those who failed treatment (N = 28, mean IC50 ± SEM = 4.53 ± 0.47 μM). The pre/post-treatment results thus showed a 2-fold difference, whereas isolated from cured versus failed patients showed a similar susceptibility, suggesting that this higher tolerance is not responsible for MIL-treatment failure. Our work highlights the need for careful monitoring of MIL susceptibility for implementation in national VL elimination programs.

Objective: To evaluate a new tool for the monitoring of Visceral Leishmaniasis (VL) treatment outcomes in primary healthcare (PHC) settings, adapted from the standardised Retrospective Quarterly Cohort Monitoring done in tuberculosis control., Methods: We developed standard case definitions for early and late VL treatment outcomes, a single register allowing for one-line entry per patient as registration tool, and quarterly reporting formats for the clinical outcomes. We pilot-tested these tools in three Indian Primary Health Centres and two Nepalese district hospitals, as well as in a charity VL treatment centre and a university hospital., Results: Data collection for early treatment outcome was easily implemented but information on late treatment outcome was hard to obtain. Effectiveness of Miltefosine under routine care conditions was about 87% at end of treatment, and 76% at 6 months post-treatment related to the high number of patients lost to follow up at the latter end point., Conclusion: A retrospective cohort monitoring methodology is conceptually a good framework for monitoring clinical outcomes for chronic conditions as VL. The monitoring of early outcomes of VL treatment is perfectly feasible in Primary Care settings. The completeness of information on late outcomes can be improved by a number of strategies that remain to be field tested. Generally, clinical outcome monitoring should be strengthened in the VL control programmes., (© 2013 John Wiley & Sons Ltd.)

Background: Miltefosine (MIL), the only oral drug for visceral leishmaniasis (VL), is currently the first-line therapy in the VL elimination program of the Indian subcontinent. Given the paucity of anti-VL drugs and the looming threat of resistance, there is an obvious need for close monitoring of clinical efficacy of MIL., Methods: In a cohort study of 120 VL patients treated with MIL in Nepal, we monitored the clinical outcomes up to 12 months after completion of therapy and explored the potential role of drug compliance, parasite drug resistance, and reinfection., Results: The initial cure rate was 95.8% (95% confidence interval [CI], 92.2-99.4) and the relapse rate at 6 and 12 months was 10.8% (95% CI, 5.2-16.4) and 20.0% (95% CI, 12.8-27.2) , respectively. No significant clinical risk factors of relapse apart from age <12 years were found. Parasite fingerprints of pretreatment and relapse bone marrow isolates within 8 patients were similar, suggesting that clinical relapses were not due to reinfection with a new strain. The mean promastigote MIL susceptibility (50% inhibitory concentration) of isolates from definite cures was similar to that of relapses. Although more tolerant strains were observed, parasite resistance, as currently measured, is thus not likely involved in MIL treatment failure. Moreover, MIL blood levels at the end of treatment were similar in cured and relapsed patients., Conclusions: Relapse in one-fifth of the MIL-treated patients observed in our study is an alarming signal for the VL elimination campaign, urging for further review and cohort monitoring.

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.)

Objective: To assess patient adherence to unsupervised single-drug miltefosine treatment for visceral leishmaniasis and to identify the factors influencing adherence., Methods: This is a prospective cohort study of 171 patients with Visceral leishmaniasis (VL) in three healthcare settings in Nepal. Adherence was assessed through pill count, checking of treatment cards and adherence questionnaires, as well as miltefosine concentration measurements at the end of treatment. Poor adherence was defined as less than 90% of required capsules taken., Results: Patient adherence to miltefosine was 83%. Predictors of adherence were being the male sex (OR = 2.60, 95% CI 1.02-6.67) and knowing the duration of treatment (OR = 3.05, 95% CI 1.16-8.04). Adherence was also better for patients who were literate and knew the side effects of treatment. Gastrointestinal side effects and negligence after the resolution of clinical symptoms of VL were the main reasons for poor adherence. Poor adherence was associated (though not statistically significant) with future relapse., Conclusion: Effective counselling during the treatment, a short take-home message on VL and on side effects and action of miltefosine, and follow-up visits are the best way to prevent poor adherence. Single end-of-treatment measurements of miltefosine concentrations as objective assessment of adherence would only be useful in addition to the subjective assessments when substantial doses of miltefosine have been missed., (© 2012 Blackwell Publishing Ltd.)

OBJECTIVE: To assess the prevalence of Post-kala-azar dermal leishmaniasis (PKDL) in 16 visceral leishmaniasis-endemic communities in Bihar, India. METHODS: Three-stage house-to-house survey of 2020 households to identify and confirm PKDL cases. RESULTS: 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 PKDL cases are discussed in detail. CONCLUSION: PKDL can develop in visceral leishmaniasis patients treated with different anti-leishmanial drugs. Migration of PKDL cases to other villages may expand visceral leishmaniasis-affected areas., (© 2012 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.

Background: Miltefosine is the only oral drug available for treatment of Indian visceral leishmaniasis (VL), which was shown to have an efficacy of 94% in a phase III trial in the Indian subcontinent. Its unrestricted use has raised concern about its continued effectiveness. This study evaluates the efficacy and safety of miltefosine for the treatment of VL after a decade of use in India., Methods: An open-label, noncomparative study was performed in which 567 patients received oral miltefosine (50 mg for patients weighing <25 kg, 100 mg in divided doses for those weighing ≥25 kg, and 2.5 mg per kg for those aged <12 years, daily for 28 days) in a directly observed manner. Patients were followed up for 6 months to see the response to therapy., Results: At the end of treatment the initial cure rate was 97.5% (intention to treat), and 6 months after the end of treatment the final cure rate was 90.3%. The overall death rate was 0.9% (5 of 567), and 2 deaths were related to drug toxicity. Gastrointestinal intolerance was frequent (64.5%). The drug was interrupted in 9 patients (1.5%) because of drug-associated adverse events., Conclusions: As compared to the phase III trial that led to registration of the drug a decade ago, there is a substantial increase in the failure rate of oral miltefosine for treatment of VL in India.

The World Health Organization (WHO) recommends several brands of long lasting insecticidal net (LN) for protection against insect vectors but also advises national programmes to monitor and evaluate performance under local conditions to help them select the most suitable LN for their setting. During the course of a community randomised trial of LNs against visceral leishmaniasis in northern India and Nepal, opportunity arose to assess the efficacy of PermaNet 2.0 (Vestergaard-Frandsen, Denmark) after two years of use against sandfly vectors. Between 63% (India) and 78% (Nepal) of LNs became holed over the course of two years, deltamethrin residues fell from 55 mg/m(2) to an average of 11.6 mg/m(2) (India) and 27.9 mg/m(2) (Nepal), but on the basis of bioassay criteria all LNs tested still met the WHO Pesticide Evaluation Scheme standard for LN effectiveness. Nets had on average only been washed 2.5 times (India) and 0.6 times (Nepal) by householders over the course of two years. The loss of insecticide was attributed to factors which had little or nothing to do with washing, such as handling, friction and torsion during daily use. Under conditions pertaining in this region of south Asia, and for two years at least, this brand of net continues to meet the criteria established by WHO for LNs., (Copyright © 2011 Royal Society of Tropical Medicine and Hygiene. Published by Elsevier Ltd. All rights reserved.)

Introduction: Post-kala-azar dermal leishmaniasis (PKDL) is a cutaneous complication appearing after treatment of visceral leishmaniasis, and PKDL patients are considered infectious to sand flies and may therefore play a role in the transmission of VL. We estimated the risk and risk factors of PKDL in patients with past VL treatment in south-eastern Nepal., Methods: Between February and May 2010 we traced all patients who had received VL treatment during 2000-2009 in five high-endemic districts and screened them for PKDL-like skin lesions. Suspected cases were referred to a tertiary care hospital for confirmation by parasitology (slit skin smear (SSS)) and/or histopathology. We calculated the risk of PKDL using Kaplan-Meier survival curves and exact logistic regression for risk factors., Results: Out of 680 past-treated VL patients, 37(5.4%) presented active skin lesions suspect of PKDL during the survey. Thirty-three of them underwent dermatological assessment, and 16 (2.4%) were ascertained as probable (2) or confirmed (14) PKDL. Survival analysis showed a 1.4% risk of PKDL within 2 years of VL treatment. All 16 had been previously treated with sodium stibogluconate (SSG) for their VL. In 5, treatment had not been completed (≤ 21 injections). Skin lesions developed after a median time interval of 23 months [interquartile range (IQR) 16-40]. We found a higher PKDL rate (29.4%) in those inadequately treated compared to those who received a full SSG course (2.0%). In the logistic regression model, unsupervised treatment [odds ratio (OR) = 8.58, 95% CI 1.21-374.77], and inadequate SSG treatment for VL in the past (OR = 11.68, 95% CI 2.71-45.47) were significantly associated with PKDL., Conclusion: The occurrence of PKDL after VL treatment in Nepal is low compared to neighboring countries. Supervised and adequate treatment of VL seems essential to reduce the risk of PKDL development and active surveillance for PKDL is needed.

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

Objective: In 2009, a random survey was conducted in Muzaffarpur district to document the clinical outcomes of visceral leishmaniasis patients (VL) treated by the public health care system in 2008, to assess the effectiveness of miltefosine against VL. We analysed the operational feasibility and cost of such periodic random surveys as compared with health facility-based routine monitoring., Methods: A random sample of 150 patients was drawn from registers kept at Primary Health Care centres. Patient records were examined, and the patients were located at their residence. Patients and physicians were interviewed with the help of two specifically designed questionnaires by a team of one supervisor, one physician and one field worker. Costs incurred during this survey were properly documented, and vehicle log books maintained for analysis., Results: Hundred and 39 (76.7%) of the patients could be located. Eleven patients were not traceable. Per patient, follow-up cost was US$ 15.51 and on average 2.27 patients could be visited per team-day. Human resource involvement constituted 75% of the total cost whereas involvement of physician costs 51% of the total cost., Conclusion: A random survey to document clinical outcomes is costly and labour intensive but gives probably the most accurate information on drug effectiveness. A health service-based retrospective cohort reporting system modelled on the monitoring system developed by tuberculosis programmes could be a better alternative. Involvement of community health workers in such monitoring would offer the additional advantage of treatment supervision and support., (© 2011 Blackwell Publishing Ltd.)

Background: 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., Methods: 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., Results: 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., Conclusions: 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.

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.

On the Indian subcontinent, visceral leishmaniasis (VL) is considered an anthroponosis. To determine possible reasons for its persistence during interepidemic periods, we mapped Leishmania infections among healthy persons and animals in an area of active VL transmission in Nepal. During 4 months (September 2007-February 2008), blood was collected from persons, goats, cows, and buffaloes in 1 village. Leishmania infections were determined by using PCR. We found infections among persons (6.1%), cows (5%), buffaloes (4%), and goats (16%). Data were georeferenced and entered into a geographic information system. The bivariate K-function results indicated spatial clustering of Leishmania spp.-positive persons and domestic animals. Classification tree analysis determined that among several possible risk factors for Leishmania infection among persons, proximity of Leishmania spp.-positive goats ranked first. Although our data do not necessarily mean that goats constitute a reservoir host of L. donovani, these observations indicate the need for further investigation of goats' possible role in VL transmission.

Objective: To document ownership and use of bednets with its determinants in the visceral leishmaniasis (VL)-endemic region where mainly non-insecticide impregnated nets are available through commercial channels, and bednets are being considered as a leishmaniasis vector control measure., Methods: In August-September 2006, semi-structured household (HH) questionnaires and observation guides were used in a random sample of 1330 HHs in VL-endemic districts of India and Nepal to collect data on VL knowledge, HH socio-economic status, bednet ownership and use patterns. An asset index was constructed to allow wealth ranking of the HH. A binary logistic response General Estimating Equations model was fitted to evaluate the determinants of bednet ownership and use., Results: The proportion of HHs with at least one bednet purchased on the commercial market was 81.5% in India and 70.2% in Nepal. The bednets were used in all seasons by 50.6% and 54.1% of the Indian and Nepalese HH owning a bed net. There was striking inequity in bednet ownership: only 38.3% of the poorest quintile in Nepal owned at least one net, compared to 89.7% of the wealthiest quintile. In India, the same trend was observed though somewhat less pronounced (73.6%vs. 93.7%). Multivariate analysis showed that poverty was an important independent predictor for not having a bednet in the HH [OR 5.39 (2.90-10.03)]., Conclusion: Given the inequity in commercial bednet ownership, free distribution of insecticide-treated bednets to the general population seems imperative to achieve a mass effect on vector density.

Objective: To compare a PCR assay and direct agglutination test (DAT) for the detection of potential markers of Leishmania infection in 231 healthy subjects living in a kala-azar endemic focus of Nepal., Methods: The sample was composed of 184 (80%) persons without any known history of KA and not living in the same house as known kala-azar cases (HNK), 24 (10%) Healthy Household Contacts (HHC) and 23 (10%) past kala-azar cases which had been successfully treated (HPK)., Results: PCR and DAT positivity scores were, respectively: HNK, 17.6% and 5.6%; HHC, 12.5% and 20.8%; HPK, 26.1% and 95.7%. The ratio PCR-positives/DAT-positives was significantly higher in HNK (ratio = 3.1) than in HHC (ratio = 0.6, P = 0.036) and in HPK (ratio = 0.2, P = 0.012). The ratio PCR-positives/DAT-positives did not significantly differ between HHC (ratio = 0.6) and HPK (ratio = 0.2, P = 0.473). The positive agreement index between PCR and DAT in HNK was 5%; in HHC, 0%; in HPK, 43%., Conclusions: Our study highlights the specific character of PCR and DAT for the exploration of Leishmania asymptomatic infections. PCR is probably more informative for very recent infections among HNK, while DAT provides more information among HHC and HPK, a feature likely related to the power of serology to track less recent infections.

Visceral leishmaniasis (VL) is a deadly vector-borne disease that causes an estimated 500 000 new cases a year. In India, Nepal and Bangladesh, VL is caused by Leishmania donovani, which is transmitted from man to man by the sandfly Phlebotomus argentipes. In 2005, these three countries signed a memorandum of understanding to eliminate VL from the region. Integrated vector management is one of the pillars of this elimination strategy, alongside early case detection and treatment. We reviewed the evidence of effectiveness of different vector control methods, to examine the potential role of insecticide treated bednets (ITNs). Indoor residual spraying has shown poor impact for various reasons and resistance to DDT is emerging in Bihar. Environmental management performed poorly compared to insecticide based methods. ITNs could give individual protection but this still needs to be proven in randomized trials. Given the constraints of indoor residual spraying, it is worthwhile to further explore the use of ITNs, in particular long lasting ITNs, as an additional tool in the VL elimination initiative.

Objective: To determine whether the use of long-lasting insecticidal nets (LLINS) at household level are effective in reducing the abundance of Phlebotomus argentipes, vector of anthroponotic visceral leishmaniasis in India, Nepal and Bangladesh., Methods: The impact of two long-lasting nets (Olyset and PermaNet) on indoor sandfly abundance was evaluated in selected houses of three endemic hamlets in Bihar (India). It was assumed that most sandflies breed inside the houses and that LLINs would progressively reduce the indoor density during the reproduction season. A campaign of indoor spraying with dichloro-diphenyl-trichloroethane (DDT) interfered with the trial but did not affect the sandfly population. Results Only the density of males of P. argentipes was significantly reduced by both the LLINs but not the females., Conclusions: These findings suggest that most female sandflies are coming from outside and that LLINs do not reduce their entry rate.

We report a case of an HIV seropositive female patient treated with a protease inhibitor-containing regimen who developed recurrent severe life-threathening episodes of haematemesis over time, caused by ruptured oesophageal varices as a consequence of a portal vein thrombosis. Coagulation tests revealed a protein S deficiency, an elevated homocysteinemia and a constitutional elevated plasma factor VIII coagulant activity. These coagulopathies and the HIV infection are independent risk factors for developing venous thromboembolic events. The protease inhibitor treatment may have played a role in increasing the thromboembolic risk. The recurrent bleedings only stopped after invasive surgery. The invasive splenorenal shunt operation was in this case a life-saving procedure.