Visceral leishmaniasis in the hills of western Nepal: A transmission assessment.

In Nepal, visceral leishmaniasis (VL) has been targeted for elimination as a public health problem by 2026. Recently, increasing numbers of VL cases have been reported from districts of doubtful endemicity including hills and mountains, threatening the ongoing VL elimination program in Nepal. We conducted a multi-disciplinary, descriptive cross-sectional survey to assess the local transmission of Leishmania donovani in seven such districts situated at altitudes of up to 1,764 meters in western Nepal from March to December 2019. House-to-house surveys were performed for socio-demographic data and data on past and current VL cases. Venous blood was collected from all consenting individuals aged ≥2 years and tested with the rK39 RDT. Blood samples were also tested with direct agglutination test, and a titer of ≥1:1600 was taken as a marker of infection. A Leishmania donovani species-specific PCR (SSU-rDNA) was performed for parasite species confirmation. We also captured sand flies using CDC light traps and mouth aspirators. The house-to-house surveys documented 28 past and six new VL cases of which 82% (28/34) were without travel exposure. Overall, 4.1% (54/1320) of healthy participants tested positive for L. donovani on at least one serological or molecular test. Among asymptomatic individuals, 17% (9/54) were household contacts of past VL cases, compared to 0.5% (6/1266) among non-infected individuals. Phlebotomus argentipes, the vector of L. donovani, was found in all districts except in Bajura. L. donovani was confirmed in two asymptomatic individuals and one pool of sand flies of Phlebotomus (Adlerius) sp. We found epidemiological and entomological evidence for local transmission of L. donovani in areas previously considered as non-endemic for VL. The national VL elimination program should revise the endemicity status of these districts and extend surveillance and control activities to curb further transmission of the disease.

The ongoing risk of Leishmania donovani transmission in eastern Nepal: an entomological investigation during the elimination era.

BACKGROUND: Visceral leishmaniasis (VL), a life-threatening neglected tropical disease, is targeted for elimination from Nepal by the year 2026. The national VL elimination program is still confronted with many challenges including the increasingly widespread distribution of the disease over the country, local resurgence and the questionable efficacy of the key vector control activities. In this study, we assessed the status and risk of Leishmania donovani transmission based on entomological indicators including seasonality, natural Leishmania infection rate and feeding behavior of vector sand flies, Phlebotomus argentipes, in three districts that had received disease control interventions in the past several years in the context of the disease elimination effort. METHODS: We selected two epidemiologically contrasting settings in each survey district, one village with and one without reported VL cases in recent years. Adult sand flies were collected using CDC light traps and mouth aspirators in each village for 12 consecutive months from July 2017 to June 2018. Leishmania infection was assessed in gravid sand flies targeting the small-subunit ribosomal RNA gene of the parasite (SSU-rRNA) and further sequenced for species identification. A segment (~ 350 bp) of the vertebrate cytochrome b (cytb) gene was amplified from blood-fed P. argentipes from dwellings shared by both humans and cattle and sequenced to identify the preferred host. RESULTS: Vector abundance varied among districts and village types and peaks were observed in June, July and September to November. The estimated Leishmania infection rate in vector sand flies was 2.2% (1.1%-3.7% at 95% credible interval) and 0.6% (0.2%-1.3% at 95% credible interval) in VL and non-VL villages respectively. The common source of blood meal was humans in both VL (52.7%) and non-VL (74.2%) villages followed by cattle. CONCLUSIONS: Our findings highlight the risk of ongoing L. donovani transmission not only in villages with VL cases but also in villages not reporting the presence of the disease over the past several years within the districts having disease elimination efforts, emphasize the remaining threats of VL re-emergence and inform the national program for critical evaluation of disease elimination strategies in Nepal.

Susceptibility status of the wild-caught Phlebotomus argentipes (Diptera: Psychodidae: Phlebotominae), the sand fly vector of visceral leishmaniasis, to different insecticides in Nepal.

BACKGROUND: Visceral leishmaniasis (VL) is targeted for elimination as a public health problem in Nepal by 2023. For nearly three decades, the core vector control intervention in Nepal has been indoor residual spraying (IRS) with pyrethroids. Considering the long-term use of pyrethroids and the possible development of resistance in the vector Phlebotomus argentipes sand flies, we monitored the susceptibility status of their field populations to the insecticides of different classes, in villages with and without IRS activities in recent years. METHODOLOGY/PRINCIPAL FINDINGS: Sand flies were collected from villages with and without IRS in five VL endemic districts from August 2019 to November 2020. The WHO susceptibility test procedure was adopted using filter papers impregnated at the discriminating concentrations of insecticides of the following classes: pyrethroids (alpha-cypermethrin 0.05%, deltamethrin 0.05%, and lambda-cyhalothrin 0.05%), carbamates (bendiocarb 0.1%) and organophosphates (malathion 5%). Pyrethroid resistance intensity bioassays with papers impregnated with 5× of the discriminating concentrations, piperonyl butoxide (PBO) synergist-pyrethroid bioassays, and DDT cross-resistance bioassays were also performed. In the IRS villages, the vector sand flies were resistant (mortality rate <90%) to alpha-cypermethrin and possibly resistant (mortality rate 90-97%) to deltamethrin and lambda-cyhalothrin, while susceptibility to these insecticides was variable in the non-IRS villages. The vector was fully susceptible to bendiocarb and malathion in all villages. A delayed knockdown time (KDT50) with pyrethroids was observed in all villages. The pyrethroid resistance intensity was low, and the susceptibility improved at 5× of the discriminating concentrations. Enhanced pyrethroid susceptibility after pre-exposure to PBO and the DDT-pyrethroid cross-resistance were evident. CONCLUSIONS/SIGNIFICANCE: Our investigation showed that P. argentipes sand flies have emerged with pyrethroid resistance, suggesting the need to switch to alternative classes of insecticides such as organophosphates for IRS. We strongly recommend the regular and systematic monitoring of insecticide resistance in sand flies to optimize the efficiency of vector control interventions to sustain VL elimination efforts in Nepal.

Susceptibility of field-collected Phlebotomus argentipes (Diptera: Psychodidae) sand flies from Bangladesh and Nepal to different insecticides.

BACKGROUND: The sand fly Phlebotomus argentipes is the vector for visceral leishmaniasis (VL) in the Indian sub-continent. In Bangladesh since 2012, indoor residual spraying (IRS) was applied in VL endemic areas using deltamethrin. In Nepal, IRS was initiated in 1992 for VL vector control using lambda-cyhalothrin. Irrational use of insecticides may lead to vector resistance but very little information on this subject is available in both countries. The objective of this study was to generate information on the susceptibility of the vector sand fly, P. argentipes to insecticide, in support of the VL elimination initiative on the Indian sub-continent. METHODS: Susceptibility tests were performed using WHO test kits following the standard procedures regarding alpha cypermethrin (0.05%), deltamethrin (0.05%), lambda-cyhalothrin (0.05%), permethrin (0.75%), malathion (5%) and bendiocarb (0.1%) in six upazilas (sub-districts) in Bangladesh. In Nepal, the tests were performed for two insecticides: alpha cypermethrin (0.05%) and deltamethrin (0.05%). Adult P. argentipes sand flies were collected in Bangladesh from six VL endemic upazilas (sub-districts) and in Nepal from three endemic districts using manual aspirators. RESULTS: The results show that VL vectors were highly susceptible to all insecticides at 60 minutes of exposure in both countries. In Bangladesh, corrected mortality was 100% at 15 minutes as well as 30 minutes of exposure. The study sites in Nepal, however, showed some diverse results, with a mortality rate less than 90% for 15 minutes of exposure with alpha cypermethrin and deltamethrin in two districts but was above 95% after 30 minutes of exposure. CONCLUSIONS: These results suggest that the insecticides tested can still be used in the national programmes of Bangladesh and Nepal. However, insecticide rotation should be performed to mitigate the possible development of insecticide resistance. Periodic susceptibility tests should be performed by the countries to get timely alerts regarding insecticide resistance.

An outbreak investigation of visceral leishmaniasis among residents of Dharan town, eastern Nepal, evidence for urban transmission of Leishmania donovani.

BACKGROUND: Visceral leishmaniasis (VL) is a predominantly rural disease, common in the low lands of eastern Nepal. Since 1997 VL cases have also been reported among residents of the city of Dharan. Our main research objective was to find out whether there had been local transmission of VL inside the city. METHODS: We conducted an outbreak investigation including a case-control study; cases were all urban residents treated for VL between 2000 and 2008 at BP Koirala Institute of Health Sciences, a university hospital in the city. For each case, we selected four random controls, with no history of previous VL; frequency-matched for age. Cases and controls were subjected to a structured interview on the main exposures of interest and potential confounders; a binominal multilevel model was used to analyze the data. We also collected entomological data from all neighborhoods of the city. RESULTS: We enrolled 115 VL patients and 448 controls. Cases were strongly clustered, 70% residing in 3 out of 19 neighborhoods. We found a strong association with socio-economic status, the poorest being most at risk. Housing was a risk factor independent from socio-economic status, most at risk were those living in thatched houses without windows. 'Sleeping upstairs' and 'sleeping on a bed' were strongly protective, OR of 0.08 and 0.25 respectively; proximity to a case was a strong risk factor (OR 3.79). Sand flies were captured in all neighborhoods; in collections from several neighborhoods presence of L. donovani could be demonstrated by PCR. CONCLUSION: The evidence found in this study is consistent with transmission of anthroponotic VL within the city. The vector P. argentipes and the parasite L. donovani have both been identified inside the town. These findings are highly relevant for policy makers; in VL endemic areas appropriate surveillance and disease control measures must be adopted not only in rural areas but in urban areas as well.

Deltamethrin and permethrin residue on long-lasting insecticidal nets after 18 months of use in a visceral leishmaniasis-endemic area in Nepal.

The insecticide residue on two types of long-lasting insecticidal nets (LLIN), Olyset Net and PermaNet 2.0, used in a visceral leishmaniasis-endemic village in eastern Nepal was quantified using HPLC. After two washes during 18 months of use the mean insecticide residues on PermaNet 2.0 and Olyset Net were 53.5 mg/m(2) (97.3% of the target dose) of deltamethrin and 911.8 mg/m(2) (91.2% of the target dose) of permethrin, respectively. These residues were close to the insecticide loads specified by the manufacturers of the two LLINs. The use of LLINs has been postulated as an alternative or complementary method to indoor residual spraying. Our results suggest that LLINs should be washed 4-5 times each year throughout their lifespan by specifically requesting villagers to wash nets on certain dates. The insecticide residue on the nets and their bioefficacy against sand fly vectors should be monitored after each wash, in various cultural settings, to assess their durability and long-term retention of the insectide.

Serological markers of sand fly exposure to evaluate insecticidal nets against visceral leishmaniasis in India and Nepal: a cluster-randomized trial.

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.

Insecticide susceptibility of Phlebotomus argentipes in visceral leishmaniasis endemic districts in India and Nepal.

OBJECTIVES: To investigate the DDT and deltamethrin susceptibility of Phlebotomus argentipes, the vector of Leishmania donovani, responsible for visceral leishmaniasis (VL), in two countries (India and Nepal) with different histories of insecticide exposure. METHODS: Standard WHO testing procedures were applied using 4% DDT and 0.05% deltamethrin impregnated papers. The effect of the physiological status (fed and unfed) of females on the outcome of the bioassays was assessed and the optimal time of exposure for deltamethrin was evaluated on a colony population. Field populations from both countries were tested. RESULTS: Fed and unfed females responded in a similar way. For exposure time on field samples 60 min was adopted for both DDT and deltamethrin. In Bihar, knockdown and mortality with DDT was respectively 20 and 43%. In Nepal almost all sand flies were killed, except at the border with Bihar (mortality 62%). With 0.05% deltamethrin, between 96 and 100% of the sand flies were killed in both regions. CONCLUSIONS: Based on literature and present data 4% DDT and 0.05% deltamethrin seem to be acceptable discriminating concentrations to separate resistant from susceptible populations. Resistance to DDT was confirmed in Bihar and in a border village of Nepal, but the sand flies were still susceptible in villages more inside Nepal where only synthetic pyrethroids are used for indoor spraying. The low effectiveness of indoor spraying with DDT in Bihar to control VL can be partially explained by this resistance hence other classes of insecticides should be tested. In both countries P. argentipes sand flies were susceptible to deltamethrin.

Development of an enzyme-linked immunosorbent assay to identify host-feeding preferences of Phlebotomus species (Diptera: Psychodidae) in endemic foci of visceral leishmaniasis in Nepal.

Anthroponotic visceral leishmaniasis, transmitted by Phlebotomus argentipes Annandale & Brunetti (Diptera: Psychodidae) sand flies, is regarded as a major problem of public health importance in the Indian subcontinent. Understanding the feeding behavior of the vector can be used to investigate changes in human-vector contact during intervention programs. An enzyme-linked immunosorbent assay (ELISA) was modified to make it suitable to identify the origin of P. argentipes and Phlebotomus papatasi Scopoli (Diptera: Psychodidae) blood meals. The sensitivity and specificity of the precipitin ring test and ELISA were compared, as well as the stability of the tests across different stages of blood meal digestion. The ELISA was more sensitive and specific than the precipitin test for identifying the sources of blood meals. When using the ELISA method with a plate reader, it was possible to obtain 100% sensitivity and specificity. When comparing the techniques across digestion stages, it was found that there was a drop in sensitivity, 48 and 72 h postblood meal for precipitin and visually read ELISA, respectively. However, the sensitivity of the ELISA using a plate reader was not altered by the digestion time. The feeding habits of P. argentipes and P. papatasi from the Terai region of Nepal, determined by the ELISA developed, showed P. papatasi to be highly anthropophilic, and P. argentipes appeared to feed both on humans and animals, in particular bovines.

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.

Effect of untreated bed nets on blood-fed Phlebotomus argentipes in kala-azar endemic foci in Nepal and India

Observational studies in the Indian subcontinent have shown that untreated nets may be protective against visceral leishmaniasis (VL). In this study, we evaluated the effect of untreated nets on the blood feeding rates of Phlebotomus argentipes as well as the human blood index (HBI) in VL endemic villages in India and Nepal. The study had a "before and after intervention" design in 58 households in six clusters. The use of untreated nets reduced the blood feeding rate by 85 percent (95 percent CI 76.5-91.1 percent) and the HBI by 42.2 percent (95 percent CI 11.1-62.5 percent). These results provide circumstantial evidence that untreated nets may provide some degree of personal protection against sand fly bites.

Natural infection of Phlebotomus argentipes with Leishmania and other trypanosomatids in a visceral leishmaniasis endemic region of Nepal.

Monitoring Leishmania infection in sand flies is important for understanding the eco-epidemiology of kala-azar and assessing the impact of the recently launched kala-azar control programme in the Indian subcontinent. We applied a PCR technique that targets rRNA genes to estimate the natural incidence of Leishmania infection in sand flies sampled in six villages of the Terai region of Nepal. Amplifications were made on 135 pools of sand flies and confirmed by sequencing. Seven pools were found to be PCR positive: in five of them we identified the rDNA signature found in Leishmania spp., whereas two other pools revealed a sequence compatible with other trypanosomatids. Different methodologies were applied to evaluate the infection rate from pools of unequal size and estimated the infection rate to range from 0.468% to 0.578% for the Leishmania group and from 0.185% to 0.279% for the non-Leishmania group. Our results highlight the diversity of flagellate infections likely to be encountered in Phlebotomus argentipes populations. Our methodology allows clear discrimination of Leishmania from other trypanosomatids and should be applied on larger insect samples or in longitudinal studies.

Effect of untreated bed nets on blood-fed Phlebotomus argentipes in kala-azar endemic foci in Nepal and India.

Observational studies in the Indian subcontinent have shown that untreated nets may be protective against visceral leishmaniasis (VL). In this study, we evaluated the effect of untreated nets on the blood feeding rates of Phlebotomus argentipes as well as the human blood index (HBI) in VL endemic villages in India and Nepal. The study had a 'before and after intervention' design in 58 households in six clusters. The use of untreated nets reduced the blood feeding rate by 85% (95% CI 76.5-91.1%) and the HBI by 42.2% (95% CI 11.1-62.5%). These results provide circumstantial evidence that untreated nets may provide some degree of personal protection against sand fly bites.

Enhanced production of antimicrobial compounds by three salt-tolerant actinobacterial strains isolated from the Sundarbans in a niche-mimic bioreactor.

A novel reactor system, the rotating disk bioreactor (RDBR), was used to mimic the niche environmental conditions of three salt-tolerant estuarine actinobacteria isolated from the Sundarbans region off the Bay of Bengal, designated MS310 (99% similar in its 16S rRNA gene sequence to Streptomyces parvallus), MS3/20 and MS1/7. The RDBR, operated at a rotational speed of one revolution per day, 50% submergence of discs, aeration rate of 1.0 vvm, and with a sucrose-containing medium, faithfully mimicked the intertidal estuarine habitat of these marine isolates, and supported biofilm formation and production of antimicrobial metabolites-in particular, actinomycin D by MS310. Onset of antibiotic production by MS310 occurs at 20 h in the RDBR compared to 55 h in a conventional stirred-tank bioreactor (STBR). Furthermore, peak antimicrobial activity is attained much earlier in the RDBR with MS310 (at 45 h) than that reported with a terrestrial strain of S. parvallus grown in a STBR (at 144 h). Peak antimicrobial activity of metabolites produced by MS1/7 and MS3/20 were also attained earlier in the RDBR (at 25 and 12 h, respectively) than in a STBR (at 80 and 28 h, respectively). Antibiotic synthesis in the three isolates, in general, appears to be associated with their growth. Overall, the RDBR may be considered the preferred alternative to the STBR for production of antimicrobials by biofilm-forming estuarine bacteria for its much higher surface/volume ratio, lower costs, and easy operability.