Basudha Khanal

Gene Expression Analysis of the Mechanism of Natural Sb(V) Resistance in Leishmania donovani Isolates from Nepal

ABSTRACT Control of visceral leishmaniasis (VL) is being challenged by the emergence of natural resistance against the first line of treatment, pentavalent antimonials [Sb(V)]. An insight into the mechanism of natural Sb(V) resistance is required for the development of efficient strategies to monitor the emergence and spreading of Sb(V) resistance in countries where VL is endemic. In this work, we have focused on the mechanism of natural Sb(V) resistance emerging in Nepal, a site where anthroponotic VL is endemic. Based on the current knowledge of Sb(V) metabolism and of the in vitro trivalent antimonial [Sb(III)] models of resistance to Leishmania spp., we selected nine genes for a comparative transcriptomic study on natural Sb(V)-resistant and -sensitive Leishmania donovani isolates. Differential gene expression patterns were observed for the genes coding for 2-thiol biosynthetic enzymes, gamma-glutamylcysteine synthetase (GCS) and ornithine decarboxylase (ODC), and for the Sb(III) transport protein aquaglyceroporin 1 (AQP1). The results indicate that the mechanism for natural Sb(V) resistance partially differs from the mechanism reported for in vitro Sb(III) resistance. More specifically, we hypothesize that natural Sb(V) resistance results from (i) a changed thiol metabolism, possibly resulting in inhibition of Sb(V) activation in amastigotes, and (ii) decreased uptake of the active drug Sb(III) by amastigotes.

Longlasting insecticidal nets for prevention of Leishmania donovani infection in India and Nepal: paired cluster randomised trial

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.

Domestic animals and epidemiology of visceral leishmaniasis, Nepal.

Proximity of Leishmania donovani–positive goats is a risk factor for human infection.

Molecular Mechanisms of Drug Resistance in Natural Leishmania Populations Vary with Genetic Background

The evolution of drug-resistance in pathogens is a major global health threat. Elucidating the molecular basis of pathogen drug-resistance has been the focus of many studies but rarely is it known whether a drug-resistance mechanism identified is universal for the studied pathogen; it has seldom been clarified whether drug-resistance mechanisms vary with the pathogens genotype. Nevertheless this is of critical importance in gaining an understanding of the complexity of this global threat and in underpinning epidemiological surveillance of pathogen drug resistance in the field. This study aimed to assess the molecular and phenotypic heterogeneity that emerges in natural parasite populations under drug treatment pressure. We studied lines of the protozoan parasite Leishmania (L.) donovani with differential susceptibility to antimonial drugs; the lines being derived from clinical isolates belonging to two distinct genetic populations that circulate in the leishmaniasis endemic region of Nepal. Parasite pathways known to be affected by antimonial drugs were characterised on five experimental levels in the lines of the two populations. Characterisation of DNA sequence, gene expression, protein expression and thiol levels revealed a number of molecular features that mark antimonial-resistant parasites in only one of the two populations studied. A final series of in vitro stress phenotyping experiments confirmed this heterogeneity amongst drug-resistant parasites from the two populations. These data provide evidence that the molecular changes associated with antimonial-resistance in natural Leishmania populations depend on the genetic background of the Leishmania population, which has resulted in a divergent set of resistance markers in the Leishmania populations. This heterogeneity of parasite adaptations provides severe challenges for the control of drug resistance in the field and the design of molecular surveillance tools for widespread applicability.

Evolutionary genomics of epidemic visceral leishmaniasis in the Indian subcontinent

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. DOI: http://dx.doi.org/10.7554/eLife.12613.001

Tinea capitis in eastern Nepal

Background  Tinea capitis is an increasing public health concern throughout the world. The clinical types and etiological agents vary from time to time and place to place. This study was undertaken to identify the etiological agents and to determine the clinico‐etiological correlation of tinea capitis in eastern Nepal.

Strong association between serological status and probability of progression to clinical visceral leishmaniasis in prospective cohort studies in India and Nepal.

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.

Metabolic adaptations of Leishmania donovani in relation to differentiation, drug resistance, and drug pressure

Antimonial (sodium stibogluconate, SSG) resistance and differentiation have been shown to be closely linked in Leishmania donovani, with SSG‐resistant strains showing an increased capacity to generate infectious (metacyclic) forms. This is the first untargeted LC‐MS metabolomics study which integrated both phenomena in one experimental design and provided insights into metabolic differences between three clinical L. donovani strains with a similar genetic background but different SSG‐susceptibilities. We performed this analysis at different stages during promastigote growth and in the absence or presence of drug pressure. When comparing SSG‐resistant and SSG‐sensitive strains, a number of metabolic changes appeared to be constitutively present in all growth stages, pointing towards a clear link with SSG‐resistance, whereas most metabolic changes were only detected in the stationary stage. These changes reflect the close intertwinement between SSG‐resistance and an increased metacyclogenesis in resistant parasites. The metabolic changes suggest that SSG‐resistant parasites have (i) an increased capacity for protection against oxidative stress; (ii) a higher fluidity of the plasma membrane; and (iii) a metabolic survival kit to better endure infection. These changes were even more pronounced in a resistant strain kept under SbIII drug pressure.

PCR and direct agglutination as Leishmania infection markers among healthy Nepalese subjects living in areas endemic for Kala-Azar.

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.

Persistence of Leishmania donovani Antibodies in Past Visceral Leishmaniasis Cases in India

ABSTRACT 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.