Visceral leishmaniasis in the Indian subcontinent: modelling epidemiology and control

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.     

Post-kala-azar dermal leishmaniasis in Nepal: a retrospective cohort study (2000-2010)

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.     

Antimonial resistance in Leishmania donovani is associated with increased in vivo parasite burden

Leishmania donovani is an intracellular protozoan parasite that causes visceral leishmaniasis (VL). Antimonials (SSG) have long been the first-line treatment against VL, but have now been replaced by miltefosine (MIL) in the Indian subcontinent due to the emergence of SSG-resistance. Our previous study hypothesised that SSG-resistant L. donovani might have increased in vivo survival skills which could affect the efficacy of other treatments such as MIL. The present study attempts to validate these hypotheses. Fourteen strains derived from Nepalese clinical isolates with documented SSG-susceptibility were infected in BALB/c mice to study their survival capacity in drug free conditions (non-treated mice) and in MIL-treated mice. SSG-resistant parasites caused a significant higher in vivo parasite load compared to SSG-sensitive parasites. However, this did not seem to affect the strains' response to MIL-treatment since parasites from both phenotypes responded equally well to in vivo MIL exposure. We conclude that there is a positive association between SSG-resistance and in vivo survival skills in our sample of L. donovani strains which could suggest a higher virulence of SSG-R strains compared to SSG-S strains. These greater in vivo survival skills of SSG-R parasites do not seem to directly affect their susceptibility to MIL. However, it cannot be excluded that repeated MIL exposure will elicit different adaptations in these SSG-R parasites with superior survival skills compared to the SSG-S parasites. Our results therefore highlight the need to closely monitor drug efficacy in the field, especially in the context of the Kala-azar elimination programme ongoing in the Indian subcontinent.     

Transmission of Leishmania donovani in the Hills of Eastern Nepal,an Outbreak Investigation in Okhaldhunga and Bhojpur Districts

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.     

Disentangling the genetic origin of Heracleum persicum (Apiaceae) in Europe: multiple introductions from multiple source populations

Biological Invasions - Unraveling the origin and colonization history of invasive plants is a long-standing challenge in evolutionary and conservation biology. The knowledge of the origin of the...     

Effect of Village-wide Use of Long-Lasting Insecticidal Nets on Visceral Leishmaniasis Vectors in India and Nepal: A Cluster Randomized Trial

Background

Visceral leishmaniasis (VL) control in the Indian subcontinent is currently based on case detection and treatment, and on vector control using indoor residual spraying (IRS). The use of long-lasting insecticidal nets (LN) has been postulated as an alternative or complement to IRS. Here we tested the impact of comprehensive distribution of LN on the density of Phlebotomus argentipes in VL-endemic villages.

Methods

A cluster-randomized controlled trial with household P. argentipes density as outcome was designed. Twelve clusters from an ongoing LN clinical trial—three intervention and three control clusters in both India and Nepal—were selected on the basis of accessibility and VL incidence. Ten houses per cluster selected on the basis of high pre-intervention P. argentipes density were monitored monthly for 12 months after distribution of LN using CDC light traps (LT) and mouth aspiration methods. Ten cattle sheds per cluster were also monitored by aspiration.

Findings

A random effect linear regression model showed that the cluster-wide distribution of LNs significantly reduced the P. argentipes density/house by 24.9% (95% CI 1.80%–42.5%) as measured by means of LTs.

Interpretation

The ongoing clinical trial, designed to measure the impact of LNs on VL incidence, will confirm whether LNs should be adopted as a control strategy in the regional VL elimination programs. The entomological evidence described here provides some evidence that LNs could be usefully deployed as part of the VL control program.

Trial registration

ClinicalTrials.gov CT-2005-015374     

New Frame Shift Mutation in Puroindoline B in Indian Wheat Cultivars Hyb65 and NI5439

Puroindoline genes pinA and pinB are the main components of the 15 kD friabilin protein reported to be associated with kernel softness. However, grain hardness of Hyb65 and NI5439, the two Indian wheat varieties, could not be explained based on the earlier identified alleles in puroindolines in wheat. Hyb65 and NI5439 are hard but based on the earlier identified allelic forms of puroindolines both the varieties could have been soft. In this investigation, puroindolines (a and b) from Hyb65 and NI5439 were characterised to understand their role in determining grain hardness. The sequence of puroindoline genes from both the varieties indicated that there was no mutation in pinA. However, there was frame shift mutation in pinB generated by insertion of a guanine residue 126 bp downstream from the start codon in both the varieties. This created new hardness allele of pinB designated as pinb-D1h. Frame shift also culminated into stop codon (TGA) 231 bp downstream from the start codon terminating protein synthesis at 77^th amino acid position. Five more stop codons (4TGA & 1TAG) were also created to the downstream positions of the first stop codon because of frame shift. There was additional point mutation in NI5439 (transition from A to G) resulting into change of amino acid residue from thymine to arginine at 205^th nucleotide position. Thus single nucleotide change in pinB resulted into truncated pin B and consequently the harder texture.     

Alteration in interactions between tumor-infiltrating lymphocytes and tumor cells in human melanomas after chemotherapy or immunotherapy

Summary Alteration in interactions between tumor-infiltrating lymphocytes (TILs) and tumor cells after chemotherapy or immunotherapy was studied in metastatic melanoma patients. Tumors were harvested from surgical specimens 17 days after the end of chemotherapy with cisplatin, vinblastine, and dacarbazine (CVD). Tumors of nonlymph-node metastases from two responders yielded neither TILs nor tumor cells, whereas those from all four nonresponders had both TILs [(1.1–13.8) × 10⁶ cells/g tumor] and tumor cells [(2.8–30.8) × 10⁶ cells/g tumor). Tumors of lymph node metastases from nine patients yielded substantial numbers both of TILs and tumor cells, regardless of different clinical responses, except with one complete responder, whose tumor did not contain tumor cells. The mean increase of TILs from these tumors (n = 14) 3–4 weeks after incubation with 200 U/ml recombinant interleukin-2 (rIL-2) was 2.5-fold, whereas there was a 56-fold increase in TILs from untreated tumors (n = 3). CD3⁺ T cells predominated in TILs before and after expansion with IL-2. IL-2-activated TILs from five of six tumors tested displayed higher cytotoxicity against autologous tumor cells than against cells from any of three allogeneic tumors. Mean tumor cell numbers (10⁶ cells/trial) obtained by serial needle biopsies for the same tumor in five patients decreased from 1.2 before therapy to 0.25 at day 4 of therapy (interferon alone), and to 0.02 at day 8 (interferon and IL-2). This decrease did not correlate with clinical responses. Yields (× 10⁶ cells/g tumor) of TILs and tumor cells in subcutaneous melanomas obtained by excisional biopsies in one nonresponder under IL-2 therapy were respectively 0.2 and 1.1 before therapy (day 0), 0.1 and <0.01 during (day 7), 0.2 and <0.01 at the end of therapy (day 21), and 0.5 and 0.5 at the time of tumor progression (day 66). Yields of TILs and tumor cells in the other nonresponder were respectively 3 and 26 before (day 0), 16 and 3 during (day 7), and 0.4 and <0.01 at the end of IL-2 therapy (day 17), and 2.5 and 6 at the time of progression (day 62). TILs in these two patients before therapy proliferated well in culture with IL-2 (570-and 720-fold, respectively), and showed higher cytotoxicity against autologous tumor cells, whereas none of those from the five tumors biopsied during or at the end of IL-2 therapy proliferated. TILs at the time of progression showed modest proliferation (54- and 76-fold, respectively) and showed major-histocompatibility-complexnonrestricted cytotoxicity. In summary, a decrease in the number of live tumor cells did not always correlate with clinical response in either therapy. CVD chemotherapy may simply impair IL-2-induced proliferation of TILs. IL-2 therapy may induce transient unresponsiveness of TILs to IL-2.This work was supported in part by grant CA 47 891 from the National Institutes of Health and a grant from the University Cancer Foundation, and Mr Richard Hunton Melanoma Found.