• Phlebotomus argentipes
• Cutaneous leishmaniasis
• Salivary antigen
• Sri Lanka
• Surveillance
• Vector exposure

• Phlebotomus/immunology
• Animals
• Sri Lanka/epidemiology
• Humans
• Biomarkers/blood
• Female
• Salivary Proteins and Peptides/immunology
• Leishmaniasis, Cutaneous/epidemiology
• Leishmaniasis, Cutaneous/immunology
• Leishmaniasis, Cutaneous/parasitology
• Leishmaniasis, Cutaneous/diagnosis
• Male
• Adult
• Leishmania donovani/immunology
• Insect Vectors/parasitology
• Insect Vectors/immunology
• Enzyme-Linked Immunosorbent Assay/methods
• Insect Proteins/immunology
• Salivary Glands/metabolism
• Middle Aged

• U01 AI136033 NIAID NIH HHS
• U01AI136033 NIH HHS
• National Institutes of Health

• Biting midges
• Diversity
• Dog shelters
• Morocco
• Mosquitoes
• Sand flies

• Animals
• Morocco/epidemiology
• Dogs
• Psychodidae/classification
• Psychodidae/parasitology
• Insect Vectors/parasitology
• Insect Vectors/classification
• Culicidae/classification
• Ceratopogonidae/classification
• Biodiversity
• Female
• Male
• Dog Diseases/parasitology
• Dog Diseases/epidemiology
• Dog Diseases/transmission
• Rural Population
• Housing, Animal
• Humans
• Zoonoses/transmission
• Zoonoses/parasitology
• Suburban Population

• Phlebotomus argentipes
• Sri Lanka
• cutaneous leishmaniasis
• salivary antigen
• surveillance
• vector exposure

• U01 AI136033 NIAID NIH HHS

• gambiense sleeping sickness
• Chagas disease
• asymptomatic infection
• modeling
• visceral leishmaniasis

• Humans
• Neglected Diseases/prevention & control
• Neglected Diseases/epidemiology
• Chagas Disease/transmission
• Chagas Disease/prevention & control
• Chagas Disease/epidemiology
• Chagas Disease/drug therapy
• Asymptomatic Infections/epidemiology
• Leishmaniasis, Visceral/prevention & control
• Leishmaniasis, Visceral/epidemiology
• Leishmaniasis, Visceral/transmission
• Leishmaniasis, Visceral/drug therapy
• Models, Theoretical
• Trypanosomiasis, African/prevention & control
• Trypanosomiasis, African/epidemiology
• Trypanosomiasis, African/transmission
• Trypanosomiasis, African/drug therapy
• India/epidemiology
• Animals

• Bill & Melinda Gates Foundation
• Bill & Melinda Gates Foundation

• Animals
• Humans
• Leishmaniasis, Visceral/epidemiology
• Nepal/epidemiology
• Cross-Sectional Studies
• Leishmania donovani/genetics
• Phlebotomus/parasitology
• Psychodidae

• 001 World Health Organization
• Instituut voor Tropische Geneeskunde

• Animals
• Humans
• Leishmaniasis, Visceral/prevention & control
• Nepal/epidemiology
• Phlebotomus
• Psychodidae
• Surveys and Questionnaires

• 001 World Health Organization

• Host preference
• Infection rate
• Leishmania donovani transmission
• Nepal
• Phlebotomus argentipes
• Seasonality
• Vector
• Visceral leishmaniasis

• Adult
• Humans
• Animals
• Cattle
• Leishmania donovani/genetics
• Nepal
• Leishmaniasis, Visceral/parasitology
• Phlebotomus/parasitology
• Psychodidae

• 716228/40/70 Instituut voor Tropische Geneeskunde

Leishmaniasis is a vector-borne parasitic disease affecting millions of people worldwide. However, in the last decade, the number of cases has been reduced from well-documented endemic parts, but sporadic cases have been reported widely from various non-endemic areas, especially from the southern Himalayan zone. This raises concerns about the emergence of new ecological niches. This warrants a critical evaluation of key factors causing this rapid spread and possibly indigenous transmission. This mini-review article is aimed to briefly address the parasite, the vector, and the environmental aspects in the transmission of leishmaniasis in these new foci against a background of worldwide endemic leishmaniasis with a special focus on the southern Himalayan zone. As the lack of knowledge about the causative parasites, vectors, reservoir hosts, atypical presentations, and their management make the problem serious and may lead to the emergence of public health issues. The present works also reviewed the existing information regarding clinical variations, diagnostic methods, treatment, its outcome, and ignite for further research in these aspects of the disease.

• Anthroponosis
• Kala azar sandfly
• Sporadic transmission
• Southern Himalaya

• Asymptomatic infection
• Global incidence
• Leishmania
• Systematic review

• Animals
• Humans
• Leishmaniasis, Visceral/diagnosis
• Leishmaniasis, Visceral/epidemiology
• Leishmaniasis, Visceral/prevention & control
• Insect Control/methods
• Nepal/epidemiology
• Phlebotomus
• Insecticides
• Psychodidae

• 001 World Health Organization

• Biogeography
• Cutaneous leishmaniasis
• Deforestation
• One health
• Stacked species distribution modelling
• Zoonosis

• Drug delivery
• Leishmaniasis
• Mannosylated thiolated nanosystem
• Nano-DDS
• Nano-adjuvants
• Nanotechnology
• Nanovaccines
• Phyto-nano-DDS
• Transferosomes

• Animals
• Antiprotozoal Agents/pharmacology
• Antiprotozoal Agents/therapeutic use
• Drug Carriers/pharmacokinetics
• Drug Delivery Systems
• Drug Therapy
• Humans
• Leishmania/drug effects
• Leishmaniasis/drug therapy
• Liposomes/therapeutic use
• Nanoparticles
• Nanotechnology/methods
• Vaccination
• Vaccines/pharmacokinetics

• 6171/Federal/ NRPU/R&D/HEC/2016 Higher Education Commission Pakistan

• HIV coinfection
• Leishmania
• Leishmaniasis
• PKDL
• drugs

Leishmaniasis is a neglected tropical disease affecting millions of people worldwide. Emerging drug resistance of Leishmania species poses threaten to the effective control and elimination programme of this neglected tropical disease.

In this work, we conducted drug-resistance testing, whole genome resequencing and proteome profiling for a recently reported clinical isolate with supposed drug resistance (HCZ), and two reference sensitive strains (DD8 and 9044) of Leishmania donovani, to explore molecular mechanisms underlying drug resistance in this parasite.

With reference to DD8 and 9044 strains, HCZ isolate showed higher-level virulence and clear resistance to antimonials in promastigote culture, infected macrophages and animal experiment. Pairwise genomic comparisons revealed genetic variations (86 copy number variations, 271 frameshift mutations in protein-coding genes and two site mutations in non-coding genes) in HCZ isolate that were absent from the reference sensitive strains. Proteomic analysis indicated different protein expression between HCZ isolate and reference strains, including 69 exclusively detected proteins and 82 consistently down-/upregulated molecules in the HCZ isolate. Integrative analysis showed linkage of 12 genomic variations (gene duplication, insertion and deletion) and their protein expression changes in HCZ isolate, which might be associated with pathogenic and antimony-resistant phenotype. Functional annotation analyses further indicated that molecules involved in nucleotide-binding, fatty acid metabolism, oxidation-reduction and transport might play a role in host-parasite interaction and drug-resistance.

This comprehensive integrative work provided novel insights into the genetic basis underlying virulence and resistance, suggesting new aspects to be investigated for a better intervention against L. donovani and associated diseases.

• Antimony resistance
• Genetic variations
• Leishmania donovani
• Proteome profiling
• Whole genome resequencing

Methods for analyzing individual-level geo-located disease data have existed for some time, but have rarely been used to analyze endemic human diseases. Here we apply such methods to nearly a decade’s worth of uniquely detailed epidemiological data on incidence of the deadly vector-borne disease visceral leishmaniasis (VL) and its secondary condition, post–kala-azar dermal leishmaniasis (PKDL), to quantify the spread of infection around cases in space and time by inferring who infected whom, and estimate the relative contribution of different infection states to transmission. Our findings highlight the key role long diagnosis delays and PKDL play in maintaining VL transmission. This detailed characterization of the spatiotemporal transmission of VL will help inform targeting of interventions around VL and PKDL cases.

Understanding of spatiotemporal transmission of infectious diseases has improved significantly in recent years. Advances in Bayesian inference methods for individual-level geo-located epidemiological data have enabled reconstruction of transmission trees and quantification of disease spread in space and time, while accounting for uncertainty in missing data. However, these methods have rarely been applied to endemic diseases or ones in which asymptomatic infection plays a role, for which additional estimation methods are required. Here, we develop such methods to analyze longitudinal incidence data on visceral leishmaniasis (VL) and its sequela, post–kala-azar dermal leishmaniasis (PKDL), in a highly endemic community in Bangladesh. Incorporating recent data on VL and PKDL infectiousness, we show that while VL cases drive transmission when incidence is high, the contribution of PKDL increases significantly as VL incidence declines (reaching 55% in this setting). Transmission is highly focal: 85% of mean distances from inferred infectors to their secondary VL cases were <300 m, and estimated average times from infector onset to secondary case infection were <4 mo for 88% of VL infectors, but up to 2.9 y for PKDL infectors. Estimated numbers of secondary cases per VL and PKDL case varied from 0 to 6 and were strongly correlated with the infector’s duration of symptoms. Counterfactual simulations suggest that prevention of PKDL could have reduced overall VL incidence by up to 25%. These results highlight the need for prompt detection and treatment of PKDL to achieve VL elimination in the Indian subcontinent and provide quantitative estimates to guide spatiotemporally targeted interventions against VL.

• Bayesian inference
• post–kala-azar dermal leishmaniasis
• spatiotemporal transmission
• transmission tree
• visceral leishmaniasis

• drug resistance
• fitness
• sand fly
• transmission
• visceral leishmaniasis

A visceral leishmaniasis outbreak was reported from a village in a low-endemic district of Bihar, India.

Outbreak investigation with house-to-house search and rapid test of kala-azar suspects and contacts was carried out. Sandfly collection and cone bio-assay was done as part of entomological study.

A spatially and temporally clustered kala-azar outbreak was found at Kosra village in Sheikhpura district with 70 cases reported till December 2018. Delay of more than a year was found between diagnosis and treatment of the index case. The southern hamlet with socio-economically disadvantaged migrant population was several times more affected than rest of the village (attack rate of 19.0% vs 0.5% respectively, OR_MH = 39.2, 95% CI 18.2–84.4). The median durations between onset of fever to first contact with any health services, onset to kala-azar diagnosis, diagnosis to treatment were 10 days (IQR 4–18), 30 days (IQR 17–73) and 1 day (IQR 0.5 to 3), respectively, for 50 kala-azar cases assessed till June 2017. Three-fourths of these kala-azar cases had out-of-pocket medical expenditure for their condition. Known risk factors for kala-azar such as illiteracy, poverty, belonging to socially disadvantaged community, migration, residing in kutcha houses, sleeping in rooms with unplastered walls and non-use of mosquito nets were present in majority of these cases. Only half the dwellings of the kala-azar cases were fully sprayed. Fully gravid female P. argentipes collected post indoor residual spraying (IRS) and low sandfly mortality on cone-bioassay indicated poor effectiveness of vector control.

There is need to focus on low-endemic areas of kala-azar. The elimination programme should implement a routine framework for kala-azar outbreak response. Complete case-finding, use of quality-compliant insecticide and coverage of all sprayable surfaces in IRS could help interrupt transmission during outbreaks.

• Epidemic
• India
• Indoor residual spraying
• Kala-azar
• Migration
• Outbreak
• Visceral leishmaniasis

The outbreaks of emerging and reemerging diseases have a high impact on the human and animal health because they are the underlying causes of disability, death, and long-term illness. For many regions those details are not, or just poorly known. Here we present on the morbidity and mortality in faunal diversities including domestic and wild species caused by various viral, bacterial, parasitic, and fungal diseases prevalent in Nepal and relevant for the wider Hindu Kush Himalaya. In addition, we provide details how antibiotic resistivity, vectors, and zoonosis have resulted on a landscape-scale in the huge public and veterinary health problem has been dealt with in the context of Nepal and the wider region.

Visceral leishmaniasis (VL) is a neglected tropical disease (NTD) caused by Leishmania protozoa that are transmitted by female sand flies. On the Indian subcontinent (ISC), VL is targeted by the World Health Organization (WHO) for elimination as a public health problem by 2020, which is defined as <1 VL case (new and relapse) per 10,000 population at district level in Nepal and sub-district level in Bangladesh and India. WHO is currently in the process of formulating 2030 targets, asking whether to maintain the 2020 target or to modify it, while adding a target of zero mortality among detected cases. The NTD Modelling Consortium has developed various mathematical VL transmission models to gain insight into the transmission dynamics of VL, identify the main knowledge gaps, and predict the feasibility of achieving and sustaining the targets by simulating the impact of varying intervention strategies. According to the models, the current target is feasible at the appropriate district/sub-district level in settings with medium VL endemicities (up to 5 reported VL cases per 10,000 population per year) prior to the start of the interventions. However, in settings with higher pre-control endemicities, additional efforts may be required. We also highlight the risk that those with post-kala-azar dermal leishmaniasis (PKDL) may pose to reaching and sustaining the VL targets, and therefore advocate adding control of PKDL cases to the new 2030 targets. Spatial analyses revealed that local hotspots with high VL incidence remain. We warn that the current target provides a perverse incentive to not detect/report cases as the target is approached, posing a risk for truly achieving elimination as a public health problem although this is taken into consideration by the WHO procedures for validation. Ongoing modelling work focuses on the risk of recrudescence when interventions are relaxed after the elimination target has been achieved.

• Control
• Elimination as a Public Health Problem
• Feasibility
• Modelling
• NTD Modelling Consortium
• Visceral leishmaniasis
• WHO guidelines

• Experimental development
• Leishmania donovani
• Sand fly

• Cutaneous Leishmaniasis
• Leishmania donovani complex
• Nepal

Post-kala-azar dermal leishmaniasis (PKDL) is a skin manifestation of visceral leishmaniasis (VL) which develops after apparent cure in some patients. PKDL is considered as the potential reservoir for the VL infection. Molecular epidemiological characterization of L. donovani isolates obtained from VL and PKDL isolates is essentially required in order to understand the transmission dynamics of the VL infection. To date, genetic variation among the VL and PKDL L. donovani isolates was not fully elucidated. Therefore, 14 clinical isolates from VL and 4 clinical isolates from PKDL were speciated by hsp70 and rDNA genes. Further characterization of L. donovani by haspB PCR demonstrates two different genotypes. All PKDL isolates have the same genetic structure. kDNA PCR-RFLP assay revealed 18 different genotypes; however, structural analysis showed the two distinct kDNA genotype population (k = 2). The kDNA fingerprint patterns of parasites from hilly districts were clustered separately from low-land districts. Therefore, further study with a large number of samples is urgently required for systematic characterization of the clinical isolates to track the molecular epidemiology of the Leishmania donovani causing VL and the role of PKDL as a reservoir.

• Differential diagnosis
• Nepal
• epidemiology
• malaria
• persistent febrile illnesses
• visceral leishmaniasis

Visceral Leishmaniasis (VL) is caused by a protozoan parasite Leishmania donovani that is transmitted to humans by an infected female sandfly, Phlebotomus argentipes. VL is common in the Indian sub-continent including Nepal and efforts for its elimination are ongoing. However, expansion of disease towards the higher altitude areas, previously considered as VL free in Nepal, may impact the ability to achieve the elimination target by 2020.

This was an exploratory study, where VL suspected patients living exclusively in the non-program districts of Nepal and presenting with fever > 2 weeks and splenomegaly was included. The patients’ blood samples were collected, and DNA was extracted. DNA was subjected to PCR amplification and subsequent sequencing. Additionally, past 10 years data of VL cases from the national databases were analysed to see the trends of the disease in program and non program districts.

Analysis of the past 10 years data revealed that trend of VL cases significantly decreased in the program districts (p = 0.001) while it increased in the non-program districts (p = 0.002). The national trend for overall incidence of VL also significantly decreased over this time period. Limited number of patients’ samples (n = 14) were subjected to molecular investigation, and four patients were found to be positive for Leishmania species by PCR. Interestingly, these cases in non-program districts were indeed also L. donovoni complex. All four patients were male with age ranges from 10 to 68 years. GenBank BLAST of the obtained DNA sequences confirmed identified specimens as L. donovani complex. We identified additional VL cases from non-program districts (including the high lands) of Nepal, indicating that the infection could be an emerging threat for the non-program areas of Nepal.

The demonstration of VL cases in areas initially considered non-endemic has raised concern about on-going transmission in those regions and may trigger subsequent government plan and action to include those areas in the elimination program. Thus, the government should consider revising the disease control programs to accommodate non-program districts for achieving the VL elimination goal set for 2020.

• High altitude
• Nepal
• Non-program districts
• Visceral leishmaniasis

• Kala-azar
• Nepal
• Népal
• comportement de recherche de santé
• delay periods
• délais de retard
• health seeking behaviour
• leishmaniose viscérale
• private sector
• report
• reporting
• secteur privé
• surveillance
• visceral leishmaniasis

• Age Distribution
• Asia, Western/epidemiology
• Female
• Humans
• Incidence
• Leishmania donovani/immunology
• Leishmania donovani/isolation & purification
• Leishmaniasis, Visceral/diagnosis
• Leishmaniasis, Visceral/epidemiology
• Leishmaniasis, Visceral/parasitology
• Leishmaniasis, Visceral/transmission
• Longitudinal Studies
• Male
• Models, Statistical
• Prevalence
• Seroepidemiologic Studies

• Bill and Melinda Gates Foundation

• Visceral leishmaniasis
• elimination
• resurgence
• surveillance

• Adolescent
• Adult
• Aged
• Aged, 80 and over
• Bangladesh
• Child
• Child, Preschool
• Disease Transmission, Infectious
• Endemic Diseases
• Epidemiologic Studies
• Female
• Geography
• Humans
• Infant
• Infant, Newborn
• Leishmaniasis, Visceral/epidemiology
• Leishmaniasis, Visceral/transmission
• Longitudinal Studies
• Male
• Middle Aged
• Models, Statistical
• Rural Population
• Spatio-Temporal Analysis
• Young Adult

• Bill and Melinda Gates Foundation

• Animals
• Antiprotozoal Agents/therapeutic use
• Coinfection/complications
• Disease Vectors
• Global Health
• HIV Infections/complications
• Humans
• Leishmania
• Leishmaniasis, Cutaneous/diagnosis
• Leishmaniasis, Cutaneous/drug therapy
• Leishmaniasis, Cutaneous/epidemiology
• Leishmaniasis, Cutaneous/physiopathology
• Leishmaniasis, Visceral/diagnosis
• Leishmaniasis, Visceral/drug therapy
• Leishmaniasis, Visceral/epidemiology
• Leishmaniasis, Visceral/physiopathology

• Asymptomatic Infections/epidemiology
• Humans
• India/epidemiology
• Leishmania/classification
• Leishmania/isolation & purification
• Leishmania/pathogenicity
• Leishmaniasis, Cutaneous/epidemiology
• Leishmaniasis, Cutaneous/pathology
• Leishmaniasis, Visceral/epidemiology
• Leishmaniasis, Visceral/pathology

• 125996 Canadian Institutes of Health Research

• new chemical entities
• treatment
• visceral leishmaniasis

• Genome diversity
• Genomics
• Leishmania donovani
• Metabolomics
• Multi-omics integration
• Virulence
• Visceral leishmaniasis

• Genome, Protozoan
• Genomics
• Humans
• India/epidemiology
• Leishmania donovani/genetics
• Leishmaniasis, Visceral/epidemiology
• Leishmaniasis, Visceral/parasitology
• Metabolomics
• Polymorphism, Single Nucleotide

• 206194 Wellcome Trust
• 206194 Wellcome Trust

• Himalayan region
• Nepal
• PCR
• Visceral leishmaniasis

The “antibiotic resistance crisis” is a major challenge for scientists and medical professionals. This steady rise in drug-resistant pathogens also extends to parasitic diseases, with antimony being the first anti-Leishmania drug that fell in the Indian subcontinent (ISC). Leishmaniasis is a major but neglected infectious disease with limited therapeutic options. Therefore, understanding how parasites became resistant to antimonials is of commanding importance. In this study, we experimentally characterized the dynamics of this resistance acquisition and show for the first time that some Leishmania populations of the ISC were preadapted to antimony resistance, likely driven by environmental factors or by drugs used in the 19th century.

Antimonials (Sb) were used for decades for chemotherapy of visceral leishmaniasis (VL). Now abandoned in the Indian subcontinent (ISC) because of Leishmania donovani resistance, this drug offers a unique model for understanding drug resistance dynamics. In a previous phylogenomic study, we found two distinct populations of L. donovani: the core group (CG) in the Gangetic plains and ISC1 in the Nepalese highlands. Sb resistance was only encountered within the CG, and a series of potential markers were identified. Here, we analyzed the development of resistance to trivalent antimonials (Sb^III) upon experimental selection in ISC1 and CG strains. We observed that (i) baseline Sb^III susceptibility of parasites was higher in ISC1 than in the CG, (ii) time to Sb^III resistance was higher for ISC1 parasites than for CG strains, and (iii) untargeted genomic and metabolomic analyses revealed molecular changes along the selection process: these were more numerous in ISC1 than in the CG. Altogether these observations led to the hypothesis that CG parasites are preadapted to Sb^III resistance. This hypothesis was experimentally confirmed by showing that only wild-type CG strains could survive a direct exposure to the maximal concentration of Sb^III. The main driver of this preadaptation was shown to be MRPA, a gene involved in Sb^III sequestration and amplified in an intrachromosomal amplicon in all CG strains characterized so far. This amplicon emerged around 1850 in the CG, well before the implementation of antimonials for VL chemotherapy, and we discuss here several hypotheses of selective pressure that could have accompanied its emergence.

IMPORTANCE The “antibiotic resistance crisis” is a major challenge for scientists and medical professionals. This steady rise in drug-resistant pathogens also extends to parasitic diseases, with antimony being the first anti-Leishmania drug that fell in the Indian subcontinent (ISC). Leishmaniasis is a major but neglected infectious disease with limited therapeutic options. Therefore, understanding how parasites became resistant to antimonials is of commanding importance. In this study, we experimentally characterized the dynamics of this resistance acquisition and show for the first time that some Leishmania populations of the ISC were preadapted to antimony resistance, likely driven by environmental factors or by drugs used in the 19th century.

• Leishmania
• antimonials
• drug resistance mechanisms
• genomics
• metabolomics

• Animals
• Disease Eradication
• Humans
• India/epidemiology
• Insect Control/methods
• Insect Vectors/parasitology
• Insect Vectors/physiology
• Insecticide-Treated Bednets
• Insecticides
• Leishmaniasis, Visceral/diagnosis
• Leishmaniasis, Visceral/drug therapy
• Leishmaniasis, Visceral/epidemiology
• Leishmaniasis, Visceral/prevention & control
• Phlebotomus/parasitology
• Phlebotomus/physiology
• Public Health Practice
• Translational Research, Biomedical
• World Health Organization

• 001 World Health Organization

We designed a straightforward method for discriminating circulating Leishmania populations in the Indian subcontinent (ISC). Research on transmission dynamics of visceral leishmaniasis (VL, or Kala-azar) was recently identified as one of the key research priorities for elimination of the disease in the ISC. VL in Bangladesh, India, and Nepal is caused by genetically homogeneous populations of Leishmania donovani parasites, transmitted by female sandflies. Classical methods to study diversity of these protozoa in other regions of the world, such as microsatellite typing, have proven of little use in the area, as they are not able to discriminate most genotypes. Recently, whole genome sequencing (WGS) so far identified 10 different populations termed ISC001-ISC010.

As an alternative to WGS for epidemiological or clinical studies, we designed assays based on PCR amplification followed by dideoxynucleotide sequencing for identification of the non-recombinant genotypes ISC001 up to ISC007. These assays were applied on 106 parasite isolates collected in Nepal between 2011 and 2014. Combined with data from WGS on strains collected in the period 2002–2011, we provide a proof-of-principle for the application of genotyping to study treatment outcome, and differential geographic distribution.

Our method can aid in epidemiological follow-up of visceral leishmaniasis in the Indian subcontinent, a necessity in the frame of the Kala-azar elimination initiative in the region.

Visceral Leishmaniasis (VL) or Kala-azar is a life-threatening neglected tropical disease that annually affects half a million people worldwide. In the Indian subcontinent (India, Nepal, Bangladesh), the disease is caused by infection with the protozoan parasite Leishmania donovani, which is transmitted by female sand flies. Currently, the Kala-azar elimination program aims at reducing the number of VL cases in the region to less than 1 in 10.000 at upazila, sub-district and district level in Bangladesh, India, and Nepal respectively. In support of this program, tools for tracking L. donovani populations are essential, because these allow monitoring geographic spread over time. However, the parasite populations in the region are highly homogeneous, requiring sequencing of the entire genome to gather sufficient information for discriminating them. Because whole genome sequencing (WGS) is impractical for large-scale use, we designed a simple alternative to identify the WGS-genotypes. Our method is based on PCR amplification followed by sequencing of one particular locus, diagnostic of each population. We provide proof-of-principle that our method can be used to track parasite populations over time, and to correlate them with clinical parameters. We believe that our assay can support the Kala-azar control efforts in the Indian subcontinent.

• Genotype
• Genotyping Techniques/methods
• Humans
• Leishmania donovani/classification
• Leishmania donovani/genetics
• Leishmania donovani/isolation & purification
• Leishmaniasis, Visceral/epidemiology
• Leishmaniasis, Visceral/parasitology
• Molecular Epidemiology/methods
• Nepal/epidemiology
• Polymerase Chain Reaction
• Sequence Analysis, DNA
• Spatio-Temporal Analysis

Visceral Leishmaniasis (VL) is a neglected tropical disease that afflicts some of the poorest populations in the world including people living in the Bihar state of India. Due to efforts from local governments, NGOs and international organizations, the number of VL cases has declined in recent years. Despite this progress, the reservoir for transmission remains to be clearly defined since it is unknown what role post kala-azar dermal leishmaniasis (PKDL) and asymptomatic infections play in transmission. This information is vital to establish effective surveillance and monitoring to sustainably eliminate VL.

We performed a longitudinal study over a 24-month period to examine VL transmission and seroconversion in households with VL, PKDL and asymptomatic infections in the Saran and Muzaffarpur districts of Bihar. During the initial screening of 5,144 people in 16 highly endemic villages, 195 cases of recently treated VL, 116 healthy rK39 positive cases and 31 PKDL cases were identified. Approximately half of the rK39-positive healthy cases identified during the initial 6-month screening period were from households (HHs) where a VL case had been identified. During the 18-month follow-up period, seroconversion of family members in the HHs with VL cases, PKDL cases, and rK39-positive individuals was similar to control HHs. Therefore, seroconversion was highest in HHs closest to the time of VL disease of a household member and there was no evidence of higher transmission in households with PKDL or healthy rK39-positive HHs. Moreover, within the PKDL HHs, (the initial 31 PKDL cases plus an additional 66 PKDL cases), there were no cases of VL identified during the initial screen or the 18-month follow-up. Notably, 23% of the PKDL cases had no prior history of VL suggesting that infection resulting directly in PKDL is more common than previously estimated.

These observations argue that acute VL cases represent the major reservoir for transmission in these villages and early identification and treatment of VL cases should remain a priority for VL elimination. We were unable to obtain evidence that transmission occurs in HHs with a PKDL case.

Visceral leishmaniasis (also known as kala-azar) caused by infection with L. donovani is a deadly parasitic disease that afflicts some of world’s poorest populations, including the people of the northern Bihar State of India. Once transmitted to a human by an infected sandfly, the L. donovani parasite migrates from the site of the sandfly bite throughout the reticuloendothelial system, resulting in high levels of infection in the spleen, liver and bone marrow that eventually lead to organ failure and death if not treated effectively. India, Nepal and Bangladesh are currently engaged in a program to eliminate visceral leishmaniasis, principally through early case detection, treatment and vector control. As humans are the only reservoir for L. donovani, it is necessary to understand how the disease is transmitted and specifically what role acute visceral leishmaniasis (VL) cases, asymptomatic infections and post kala-azar dermal leishmaniasis (PKDL) cases play in transmission. We therefore performed a study to determine seroconversion for antibodies against the L. donovani rK39 antigen as a surrogate for transmission in households with VL cases, asymptomatic infections and PKDL cases in 16 highly endemic villages over a 2-year period in Bihar, India. We observed that most transmission occurred in the VL households and further that it occurred closest to the time of acute disease. We were unable to confirm that transmission occurred in the households with either asymptomatic infections or PKDL cases. These observations argue that active surveillance to diagnose and treat VL cases as soon as possible to reduce transmission should remain a priority for VL elimination.

• Adolescent
• Adult
• Antiprotozoal Agents/therapeutic use
• Asymptomatic Infections/epidemiology
• Child
• Child, Preschool
• Disease Reservoirs/parasitology
• Family Characteristics
• Female
• Humans
• India/epidemiology
• Infant
• Leishmaniasis, Cutaneous/complications
• Leishmaniasis, Cutaneous/drug therapy
• Leishmaniasis, Cutaneous/transmission
• Leishmaniasis, Visceral/complications
• Leishmaniasis, Visceral/epidemiology
• Leishmaniasis, Visceral/parasitology
• Leishmaniasis, Visceral/transmission
• Longitudinal Studies
• Male
• Middle Aged
• Neglected Diseases/epidemiology
• Neglected Diseases/parasitology
• Seroconversion
• Young Adult

• Grand Challenges Canada

•

Three systems of loop-mediated isothermal amplification (LAMP) were developed for diagnosing leishmaniasis.

Visceral leishmaniasis (VL), one of the most important neglected tropical diseases, is caused by Leishmania donovani eukaryotic protozoan parasite of the genus Leishmania, the disease is prevalent mainly in the Indian sub-continent, East Africa and Brazil. VL can be diagnosed by PCR amplifying ITS1 and/or kDNA genes. The current study involved the optimization of Loop-mediated isothermal amplification (LAMP) for the detection of Leishmania DNA in human blood or tissue samples. Three LAMP systems were developed; in two of those the primers were designed based on shared regions of the ITS1 gene among different Leishmania species, while the primers for the third LAMP system were derived from a newly identified repeated region in the Leishmania genome. The LAMP tests were shown to be sufficiently sensitive to detect 0.1 pg of DNA from most Leishmania species. The green nucleic acid stain SYTO16, was used here for the first time to allow real-time monitoring of LAMP amplification. The advantage of real time-LAMP using SYTO 16 over end-point LAMP product detection is discussed. The efficacy of the real time-LAMP tests for detecting Leishmania DNA in dried blood samples from volunteers living in endemic areas, was compared with that of qRT-kDNA PCR.

• Isothermal loop-mediated amplification
• Leishmania donovani
• Real time LAMP
• SYTO16
• Visceral leishmaniasis

As Bangladesh, India and Nepal progress towards visceral leishmaniasis (VL) elimination, it is important to understand the role of asymptomatic Leishmania infection (ALI), VL treatment relapse and post kala-azar dermal leishmaniasis (PKDL) in transmission.

We reviewed evidence systematically on ALI, relapse and PKDL. We searched multiple databases to include studies on burden, risk factors, biomarkers, natural history, and infectiveness of ALI, PKDL and relapse. After screening 292 papers, 98 were included covering the years 1942 through 2016. ALI, PKDL and relapse studies lacked a reference standard and appropriate biomarker. The prevalence of ALI was 4–17-fold that of VL. The risk of ALI was higher in VL case contacts. Most infections remained asymptomatic or resolved spontaneously. The proportion of ALI that progressed to VL disease within a year was 1.5–23%, and was higher amongst those with high antibody titres. The natural history of PKDL showed variability; 3.8–28.6% had no past history of VL treatment. The infectiveness of PKDL was 32–53%. The risk of VL relapse was higher with HIV co-infection. Modelling studies predicted a range of scenarios. One model predicted VL elimination was unlikely in the long term with early diagnosis. Another model estimated that ALI contributed to 82% of the overall transmission, VL to 10% and PKDL to 8%. Another model predicted that VL cases were the main driver for transmission. Different models predicted VL elimination if the sandfly density was reduced by 67% by killing the sandfly or by 79% by reducing their breeding sites, or with 4–6y of optimal IRS or 10y of sub-optimal IRS and only in low endemic setting.

There is a need for xenodiagnostic and longitudinal studies to understand the potential of ALI and PKDL as reservoirs of infection.

The role of asymptomatic Leishmania infection (ALI), PKDL and VL relapse in transmission is unclear as VL elimination is achieved in the Indian subcontinent. ALI, PKDL and relapse studies lacked a reference standard and appropriate biomarker. ALI was 4–17-fold more prevalent than VL. The risk of ALI was higher in VL case contacts. Most infections remained asymptomatic or resolved spontaneously. The natural history of PKDL showed variability. Twenty nine percent had no past history of VL treatment. The risk of VL relapse was higher with HIV co-infection. Modelling studies predicted different effects. Early diagnosis was unlikely to eliminate VL in the long term. ALI was predicted to contribute to 82% of the overall transmission, VL to 10% and PKDL to 8%. Another model predicted that VL cases were the main driver for transmission. VL elimination was predicted if the sandfly density was reduced by 67% by killing the sandfly or by 79% by reducing their breeding sites, or with 4–6y of optimal IRS or 10y of sub-optimal IRS and only in low endemic setting. There is a need for more studies to fully understand the potential of ALI and PKDL as reservoirs of infection.

Visceral leishmaniasis (VL) is a neglected tropical disease transmitted by sandflies. On the Indian subcontinent (ISC), VL is targeted for elimination as a public health problem by 2017. In the context of VL, the elimination target is defined as an annual VL incidence of <1 per 10,000 capita at (sub-)district level. Interventions focus on vector control, surveillance and on diagnosing and treating VL cases. Many endemic areas have not yet achieved optimal control due to logistical, biological as well as technical challenges. We used mathematical modelling to quantify VL transmission dynamics and predict the feasibility of achieving the VL elimination target with current control strategies under varying assumptions about the reservoir of infection in humans.

We developed three deterministic age-structured transmission models with different main reservoirs of infection in humans: asymptomatic infections (model 1), reactivation of infection after initial infection (model 2), and post kala-azar dermal leishmaniasis (PKDL; model 3). For each model, we defined four sub-variants based on different assumptions about the duration of immunity and age-patterns in exposure to sandflies. All 12 model sub-variants were fitted to data from the KalaNet study in Bihar (India) and Nepal, and the best sub-variant was selected per model. Predictions were made for optimal and sub-optimal indoor residual spraying (IRS) effectiveness for three different levels of VL endemicity.

Structurally different models explained the KalaNet data equally well. However, the predicted impact of IRS varied substantially between models, such that a conclusion about reaching the VL elimination targets for the ISC heavily depends on assumptions about the main reservoir of infection in humans: asymptomatic cases, recovered (immune) individuals that reactivate, or PKDL cases.

Available data on the impact of IRS so far suggest one model is probably closest to reality (model 1). According to this model, elimination of VL (incidence of <1 per 10,000) by 2017 is only feasible in low and medium endemic settings with optimal IRS. In highly endemic settings and settings with sub-optimal IRS, additional interventions will be required.

The online version of this article (doi:10.1186/s13071-016-1292-0) contains supplementary material, which is available to authorized users.