Lalita Roy

Background: Vector-borne diseases (VBDs), including malaria, visceral leishmaniasis, lymphatic filariasis, Japanese encephalitis, and dengue, are major public health concerns and are either slated for elimination or projected for control in Nepal. One of the major challenges in controlling these VBDs is halting their emergence and expansion in diverse geo-ecological and climatic regions. In this study, we collected vectors of major VBDs to assess their distribution, diversity, and associations with ecological variations and to provide an updated understanding of the current situation. Methodology/principal findings: A descriptive cross-sectional survey was conducted in five districts of Koshi Province, eastern Nepal, during May and June 2023 to collect vectors across three distinct geo-ecological and climatic regions: mountains, hills, and lowlands, situated at altitudes ranging from 98 to 1,274 meters. Adult mosquitoes and sand flies were captured using CDC miniature light traps, BG-Sentinel traps, and manual aspirators. We fitted generalized linear models (GLM) with a negative binomial distribution to assess the association between vector abundance and geo-ecological and climatic variables for two vector species ( Culex quinquefasciatus and Phlebotomus argentipes ). We found the malaria vector, Anopheles annularis , the lymphatic filariasis vector, Cx. quinquefasciatus and the visceral leishmaniasis vector, Ph. argentipes , across all three geo-ecological regions. Other vectors of the malaria parasite, An. pseudowillmori and An. willmori , and Japanese encephalitis vector Cx. tritaeniorhynchus were recorded only in hilly districts. Mean temperature and rainfall had a positive effect on Cx. quinquefasciatus density, but a deleterious effect on Ph. argentipes. Culex quinquefasciatus and Ph. argentipes were captured in higher abundance at the household level in the hills (IRR = 1.23 and IRR = 13.00, respectively) and mountains (IRR = 1.96 and IRR = 4.00, respectively) compared with the lowlands. Conclusion: Two major vectors, Cx. quinquefasciatus and Ph. argentipes were indiscriminately present in all geo-ecological regions. Climatic variables seemed conducive to vector survival, distribution, and growth across diverse altitudes from the lowlands to the high hills and mountains. Our findings highlight the need for the VBDs control programme to implement regular monitoring, strengthen existing surveillance systems, and support evidence-based planning and implementation of vector control interventions across wider geo-ecological regions to prevent disease transmission.

This article provides a comprehensive guide for the processing and mounting of phlebotomine sand fly specimens, which is crucial for species identification and pathogen detection and isolation. It discusses a range of techniques suitable for both field and laboratory settings. The guide includes detailed instructions on sand fly collection, handling, covering, and euthanasia (recommending dry freezing or CO ₂ over chemicals) as well as conservation strategies, such as cold storage and preservation in ethanol. The quality of preparation of certain anatomical structures (genital organs, head and wings) is essential for their proper microscopic observation and is described in this work. The article also presents detailed sample processing, including the clearing process with agents such as potassium hydroxide then Marc-André solution. The mounting process compares different media, emphasizing their optical properties and preservation potential. Hoyer fluid (also known as chloral gum) is recommended for quick observation, particularly for spermathecae, due to its clarity, although it is not suitable for long-term storage. Other media discussed include polyvinyl alcohol, Euparal ^® (for limited water tolerance), and Canada balsam (a hydrocarbon-soluble medium), with the latter two offering long-term preservation capabilities. Innovative molecular biology approaches such as DNA sequencing and MALDI-ToF, which require particular attention to sample processing, are also addressed. Furthermore, short video clips illustrating various mounting techniques as well as translations in many different languages are provided, allowing the guideline to reach the diverse needs and expectations of the global scientific community.

Nepal is committed to eliminating visceral leishmaniasis as a public health problem by 2030. The scattered distribution of VL cases across wide geo-ecological regions, including areas previously considered unsuitable for the survival of vectors and the transmission of the pathogen, poses a major threat to Nepal’s national VL elimination programme. Regular monitoring and accurate identification of sand fly species are essential for implementing tailored vector control interventions. Hence, this study aimed to update the distribution of sand fly species with a focus on Leishmania vectors and evaluate DNA barcoding as a complementary tool for their identification. Sand flies were collected from 43 districts with active VL cases across the country between 2017 and 2022. The mitochondrial COI gene was amplified for DNA barcoding analysis. The primary vector, Phlebotomus argentipes, was present in all except three districts. Potential vectors, Ph. (Adlerius) spp. and Ph. major, were found common in high-altitude regions. The species identification success rate of generated COI barcode sequences based on the “Best Close Match” was 97%, indicating high accuracy in delineating sand flies to the species level. The information on the distribution of phlebotomine sand flies and the potential use of DNA barcoding for their identification could be milestones for sand fly research and help to guide the vector control interventions in support of VL elimination in Nepal.

Background: Vector-borne diseases, being major public health concerns, are either slated for elimination or projected for control in Nepal. One of the major challenges in controlling these VBDs is to halt their emergence and expansion in diverse geo-ecological and climatic regions. In this study, we collected vectors of major VBDs to understand their distribution, diversity, and association with ecological variances. Methodology/Principal findings: A descriptive cross-sectional survey was conducted to collect the vectors in five districts situated at altitudes of up to 98 – 1,274 meters in Koshi Province, Eastern Nepal, representing three distinct geo-ecological and climatic regions: mountain, hills and lowlands during May and June 2023. Adult vectors were captured using CDC miniature light traps, BG sentinel traps, and manual aspirators. Association of vectors’ abundance in function of the geo-ecological and climatic variables was assessed in two vector species ( Culex quinquefasciatus and Phlebotomus argentipes ). We found the malaria vector Anopheles annularis , the lymphatic filariasis vector Cu. quinquefasciatus and visceral leishmaniasis vector Ph. argentipes from all three geo-ecological regions. The other vectors of the malaria parasite, An. pseudowillmori and An. willmori , and Japanese encephalitis vector Cu. tritaeniorhynchus were recorded from hilly districts only. Mean temperature and rainfall had positive effect on Cu. quinquefasciatus density and deleterious on Ph. argentipes. Culex quinquefasciatus and Ph. argentipes were captured in high abundance per household in hills (IRR = 1.23 and IRR = 13.00, respectively) and mountain (IRR = 1.96 and IRR = 4.00, respectively) as compared to lowland. Conclusion: Two major vectors, Cu. quinquefasciatus and Ph. argentipes were indiscriminately present in all geo-ecological regions. Climatic variables favour vectors’ survival, distribution and growth in diverse altitudes, including areas previously impervious to the vectors and VBDs. Our findings alert the VBD control program and suggest regular monitoring, strengthening the existing surveillance system and evidence-based planning and implementation of vector control interventions in wider geo-ecological regions to prevent the disease transmission. Authors Summary: The Government of Nepal is committed to eliminate and control major vector-borne diseases: malaria, visceral leishmaniasis, lymphatic filariasis, Japanese encephalitis and dengue as a public health problem by 2030. These diseases, once endemic to lowlands with tropical and subtropical climates only, have been spread to a wide geography, including hills and mountains. These high-altitude areas were considered unsuitable for the existence of vectors and transmission of pathogens. Considering the impact of climate change on founding a favorable environment for the establishment of viable populations of vectors for pathogens transmission and thus threatening all the efforts of VBDs control and elimination, we conducted a cross-sectional survey to explore the diversity of vectors in varied geo-ecological regions. We collected vectors from five districts situated at different altitudes and having fairly diverse geography. Major vectors of responsible of transmission of pathogens causing VL and LF were abundantly present in all geo-ecological regions, while vectors of malarial parasites and dengue virus were sparsely present in fewer areas. This study also examined the feasibility of the integrated vector survey in favour of maximising the available resources for entomological investigations. Further, this study provides updated information on the diversity of the vectors in the areas having cases of VBDs and suggests authorities for regular monitoring, strengthening the existing surveillance system and evidence-based planning and implementation of vector control interventions to prevent the disease transmission in wider geo-ecological regions.

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.

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. Graphical Abstract:

Background Visceral leishmaniasis (VL), a life-threatening neglected tropical disease, is targeted for elimination from Nepal by the year 2026. The national VL elimination programme 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. 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 VL endemic districts in two epidemiologically contrasting settings; each district with one village with and one without reported VL cases in recent years.Methods 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. Sand fly species level identification was based on morphological characters and verified by the DNA barcoding method. We assessed P. argentipes abundance and seasonality based on the light trap collections, considering it as the standard method of collection. 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 in sand fly density was observed in June-July and September-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, 52.7% in VL villages and 74.2% in non-VL villages, followed by cattle.Conclusion Our findings highlight the risk of ongoing L. donovani transmission in VL endemic districts even in villages not reporting the presence of the disease over the past many years and emphasize the remaining threats to the VL elimination programme 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 vectorPhlebotomus argentipessand 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 50) 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 thatP.argentipessand 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.

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 possible development of resistance of the vector Phlebotomus argentipes sand flies, we monitored 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 50 ) 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 for 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. Author summary: Visceral leishmaniasis (VL), transmitted by P. argentipes sand flies, is endemic in South-East Asian countries such as Bangladesh, India and Nepal, and is on the verge of elimination as a public health problem in Nepal by 2023. As part of the WHO Global Vector Control Response, entomological surveillance including insecticide resistance monitoring is one of the four main pillars of this strategy. In the early 1990s, the historical use of DDT for sand fly vector control was replaced with deltamethrin or alpha-cypermethrin, which have now been in use for almost three decades in Nepal. Suspecting that this long-term use of pyrethroids might have selected resistance in sand fly populations which would jeopardize control efforts, we conducted the first comprehensive survey to generate contemporary evidence of insecticide resistance in Nepal. For this, we performed WHO susceptibility tests in five VL endemic districts and found strong evidence of pyrethroid resistance in vector populations from the areas receiving IRS. Resistance mechanisms involved would probably be kdr mutations and monooxygenase. This study also endorses regular insecticide resistance monitoring to inform evidence-based decisions on insecticide use for vector control and to maintain the effectiveness of vector control measures as a core intervention in the fight against VL.

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. 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. 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. 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.