Acute encephalitis syndrome (AES) is a major public health concern in India, especially in Muzaffarpur, Bihar, as outbreaks of this neurological disorder have brought on significant morbidity and mortality. It is usually characterized by sudden fever, seizures, confusion, and altered mental status in children along with infectious agents, environmental factors, and malnutrition. It is evident during litchi harvest (April-June), affecting malnourished children from poor families. Among 622 AES cases from May till July 2019; a significant majority were found to have higher mortality and morbidity indices among female Scheduled Caste children. Access barriers in the social and cultural context delayed health care in the event of morbidity. Earlier speculation about associating litchi intake was set aside as most of affected children stayed far from orchards. Clinically, it manifests with fever starting suddenly associated with seizures, altered sensorium, hypoglycemia, hyponatremia, and elevated biomarkers CPK, LDH, and ammonia. High humidity and inadequate mosquito net use are linked. However, these observational studies could not show laboratory evidence of vectors of Japanese encephalitis. Priorities in action include strengthening surveillance, nutrition, education, and better health care access.

Abstract Background Vector control based on indoor residual spraying (IRS) is one of the main components of the visceral leishmaniasis (VL) elimination programme in India. Dichlorodiphenyltrichloroethane (DDT) was used for IRS until 2015 and was later replaced by the synthetic pyrethroid alpha-cypermethrin. Both classes of insecticides share the same target site, the voltage-gated sodium channel (Vgsc). As high levels of resistance to DDT have been documented in the local sand fly vector, Phlebotomus argentipes, it is possible that mutations in the Vgsc gene could provide resistance to alpha-cypermethrin, affecting current IRS pyrethroid-based vector control. Methods This study aimed to compare frequencies of knockdown resistance (kdr) mutations in Vgsc between two sprayed and two unsprayed villages in Bihar state, India, which had the highest VL burden of the four endemic states. Across four villages, 350 female P. argentipes were collected as part of a 2019 molecular xenomonitoring study. DNA was extracted and used for sequence analysis of the IIS6 fragment of the Vgsc gene to assess the presence of kdr mutations. Results Mutations were identified at various positions, most frequently at codon 1014, a common site known to be associated with insecticide resistance in mosquitoes and sand flies. Significant inter-village variation was observed, with sand flies from Dharampur, an unsprayed village, showing a significantly higher proportion of wild-type alleles (55.8%) compared with the three other villages (8.5–14.3%). The allele differences observed across the four villages may result from selection pressure caused by previous exposure to DDT. Conclusions While DDT resistance has been reported in Bihar, P. argentipes is still susceptible to pyrethroids. However, the presence of kdr mutations in sand flies could present a threat to IRS used for VL control in endemic villages in India. Continuous surveillance of vector bionomics and insecticide resistance, using bioassays and target genotyping, is required to inform India’s vector control strategies and to ensure the VL elimination target is reached and sustained. Graphical Abstract

Background: Lotus (Nelumbo nucifera Gaertn.) is a perennial rhizomatous aquatic plant naturally growing in India from Kashmir to Kanyakumari with enormous phenotypic diversity in shape, size and colour. It serves as a gene pool for breeding new, improved cultivars; nevertheless, plants’ natural habitat is disappearing daily due to rapid urbanization driven by population growth. Therefore, the current study was formulated to estimate the genetic diversity for upcoming breeding initiatives and further conservation of available germplasm of lotus. Methods: In the current study, in-situ morphological observations were recorded from thirty-three lotus genotypes between 2022 and 2023, from different locations of Bihar (India). Morphological data on 21 quantitative traits were recorded from selected natural lotus growing site with five replications. Result: Based on principal component analysis, six principal components were identified, which accounted for 88.97% of total genetic variation. In D2 analysis, genotypes were categorized into six different clusters. Cluster II consisted of the maximum number of genotypes (23) followed by cluster I (4). The highest intra-cluster distance was recorded for cluster II (184.40), while the inter-cluster distance was highest between cluster VI and III (5098.07). The study highlights the enormous genetic diversity among the genotypes of lotus and generates information about suitable breeding material(s) for future crop improvement programs. The genotypes from distant clusters may serve as potential genetic material for further selection or a superior heterotic combination for lotus breeding.

BackgroundThe kala-azar elimination programme has resulted in a significant reduction in visceral leishmaniasis (VL) cases across the Indian Subcontinent. To detect any resurgence of transmission, a sensitive cost-effective surveillance system is required. Molecular xenomonitoring (MX), detection of pathogen DNA/RNA in vectors, provides a proxy of human infection in the lymphatic filariasis elimination programme. To determine whether MX can be used for VL surveillance in a low transmission setting, large numbers of the sand fly vector Phlebotomus argentipes are required. This study will determine the best method for capturing P. argentipes females for MX.Methodology/principal findingsThe field study was performed in two programmatic and two non-programmatic villages in Bihar, India. A total of 48 households (12/village) were recruited. Centers for Disease Control and Prevention light traps (CDC-LTs) were compared with Improved Prokopack (PKP) and mechanical vacuum aspirators (MVA) using standardised methods. Four 12x12 Latin squares, 576 collections, were attempted (12/house, 144/village,192/method). Molecular analyses of collections were conducted to confirm identification of P. argentipes and to detect human and Leishmania DNA. Operational factors, such as time burden, acceptance to householders and RNA preservation, were also considered. A total of 562 collections (97.7%) were completed with 6,809 sand flies captured. Females comprised 49.0% of captures, of which 1,934 (57.9%) were identified as P. argentipes. CDC-LTs collected 4.04 times more P. argentipes females than MVA and 3.62 times more than PKP (p<0.0001 for each). Of 21,735 mosquitoes in the same collections, no significant differences between collection methods were observed. CDC-LTs took less time to install and collect than to perform aspirations and their greater yield compensated for increased sorting time. No significant differences in Leishmania RNA detection and quantitation between methods were observed in experimentally infected sand flies maintained in conditions simulating field conditions. CDC-LTs were favoured by householders.Conclusions/significanceCDC-LTs are the most useful collection tool of those tested for MX surveillance since they collected higher numbers of P. argentipes females without compromising mosquito captures or the preservation of RNA. However, capture rates are still low.

Visceral leishmaniasis (VL) is declining in India and the World Health Organization's (WHO) 2020 'elimination as a public health problem' target has nearly been achieved. Intensified combined interventions might help reach elimination, but their impact has not been assessed. WHO's Neglected Tropical Diseases 2021–2030 roadmap provides an opportunity to revisit VL control strategies. We estimated the combined effect of a district-wide pilot of intensified interventions in the highly-endemic Vaishali district, where cases fell from 3,598 in 2012–2014 to 762 in 2015–2017. The intensified control approach comprised indoor residual spraying with improved supervision; VL-specific training for accredited social health activists to reduce onset-to-diagnosis time; and increased Information Education & Communication activities in the community. We compared the rate of incidence decrease in Vaishali to other districts in Bihar state via an interrupted time series analysis with a spatiotemporal model informed by previous VL epidemiological estimates. Changes in Vaishali's rank among Bihar's endemic districts in terms of monthly incidence showed a change pre-pilot (3rd highest out of 33 reporting districts) vs. during the pilot (9th) (p<1e-10). The rate of decline in Vaishali's incidence saw no change in rank at 11th highest, both pre-pilot & during the pilot. Counterfactual model simulations suggest an estimated median of 352 cases (IQR 234–477) were averted by the Vaishali pilot between January 2015 and December 2017, which was robust to modest changes in the onset-to-diagnosis distribution. Strengthening control strategies may have precipitated a substantial change in VL incidence in Vaishali and suggests this approach should be piloted in other highly-endemic districts.

Female sand flies Phlebotomus argentipes are well-established pathogen transmitter for Visceral Leishmaniasis (VL) among humans and Dichlorodiphenyltrichloroethane (DDT) served as an important tool for controlling their population.After the decades of DDT use, P. argentipes has developed resistance against it affecting every aspect of vector control at grass-root level.In a way of exploring new 'strategic weapon' against sand flies, Novalurone -a noble Insect Growth Regulators (IGRs), inhibiting life-cycle and checking insect population at their non-infective stage has been evaluated for the first time ever against the laboratory colonized DDT resistant sand flies.It was observed that DDT resistant sand flies tolerating insecticide pressure successfully exhibited susceptibility at highest concentration of Novalurone processed larval diet (i.e., at 150000 ppm) following slowed growth rate that doesn't allowed them to metamorphed into the pupal stage.Moreover, their survival (1.8±94.50)was observed to be lasted till 2 nd stage only in which they attained absolute mortality.These preliminary laboratory based results certifies Novalurone to be very much effective for nailing effective checks upon natural growth process of immature DDT resistant sand flies and hence could be incorporated as an important preposition as alone or as a synergist with insecticide for total and long-lasting control over the VL vector population.

BACKGROUND:Visceral leishmaniasis (VL) or Kala-azar has been a major public health problem in Bihar, India, for several decades. A few VL infected districts including Vaishali have reported >600 cases annually. Hence, in 2015, the Government of India entrusted ICMR-Rajendra Memorial Research Institute of Medical Sciences, Patna, to implement an integrated control strategy for achieving the VL elimination target (<1 case per 10,000 people at the block level) in the Vaishali District of Bihar.METHODOLOGY:This study was conducted between January 2015 and December 2016. An integrated control strategy including the spatio-temporal mapping of VL-case distribution, active case detection, chemical-based vector control using indoor residual spraying (IRS), community awareness campaigns, the training of IRS members, the training of medical doctors for effective treatment, daily monitoring and the supervision of IRS activities, logistic management, post-IRS quality assurance, epidemiological surveillance, and entomological monitoring was performed. An insecticide quantification test was performed for evaluating the IRS quality on sprayed walls. A modern compression pump was used to maintain spray quality on different wall surfaces. The impact of IRS was assessed through sand fly collection in human dwellings and cattle sheds in pre- and post-IRS. The insecticide susceptibility of local P. argentipes was performed before each IRS round (in February and June) during 2015-2016. Statistical analysis such as the mean, percentage, and 95% CI were used to summarize the results.FINDINGS:All 16 blocks of the Vaishali District achieved the VL elimination target in 2016. The integrated VL control strategy helped reduce the number of VL cases from 664 in 2014 to 163 in 2016 and the number of endemic villages from 282 in 2014 to 142 in 2016. The case reduction rate was increased from 22.6% in 2014 to 58.8% in 2016. On average, 74 VL infected villages became Kala-azar free each year from 2015 to 2016.CONCLUSIONS:The results of this study suggest that the elimination of VL is possible from all endemic blocks of Bihar if the integrated Vaishali VL control strategy is applied under strong monitoring and supervision.

Background: After the decades of Dichlorodiphenyltrichloroethane (DDT) use, Phlebotomus argentipes reportedly developed resistance against it affecting every aspect of vector control at grass-root level. Although DDT based Indoor Residual Spray (IRS) has been replaced with Alphacypermethrine-a Synthetic Pyrethroid (SP) based insecticide, since 2016 but its successful implementation at the Visceral Leishmaniasis (VL) endemic regime of Bihar doesn’t cause much effect upon VL vector density. Furthermore, the outcomes of existing operational research works, it had been observed that VL vectors are continuously changing its behavior under the pressure of insecticides. Methods: For validating the hypothesis, present study has been carried out at Vaishali and Patna being highly and semi-endemic sites respectively for quantifying the oriental behavior among VL vectors persuaded by the IRS and enforce them to remain alive and get trapped in light trap even after changed chemical composition of IRS i.e., SP-IRS from routine DDT-IRS. Results: Following results, a significant reduction in sand fly density (i.e., 33.09% and 29.16%) was observed for outdoor and indoor caught sand flies, collected with light trap and aspirator respectively. Significant higher no. of sand fly collection in terms of per light traps per night was recorded from the outdoor sites than those from indoor habitat for each village of Vaishali and Saran district of Bihar. Higher no. of male sand flies than to that of female ones were collected from outdoor sites and only unfed female sand flies (i.e., 100%) were caught following SP-IRS from each study villages of Vaishali and Saran districts of Bihar. Conclusions: The results of higher no. of sand flies collection from the outdoor sites as compared with the indoor habitat validate the hypothesis of gradual shifting of habitat of VL vectors from endophilic to exophilic which is undoubtedly followed due to the fact of developed resistance among them against chemical constituent of IRS. Results provide very useful information about the sand fly dynamics under the impact of IRS and accordingly, advocates the combined approach of IRS along with insecticidal fogging together at a same time that could be an effective dividend for maximum VL vector control along for negotiating VL cases at par for longer duration during the maintenance phase at the VL foci.

Vermicompost is an organic manure (bio-fertilizer) produced as the vermicast by earth worm feeding on biological waste material; plant residues. Vermi casts are popularly called as ‘Black Gold’. This compost is an odorless, clean, organic material containing adequate quantities of N, P, K and several micronutrients essential for plant growth. The prime market for vermicompost is in agriculture and horticulture. Farmers are already using vermicompost in large quantities. Small and marginal farmers would do well to produce vermicompost on their own. Home gardens, kitchen gardens and commercial plant nurseries are an excellent market for vermicompost in urban areas. This enterprise offers tremendous scope for expansion in rural areas because it has the potential to engage small farmers and women who can easily set up such units at the household level. The enterprise is based on the conversion of waste matter into rich plant humus by using earthworms. No specific skill is required. It is a viable production oriented enterprise with tremendous scope for expansion in rural areas. Moreover, it will solve the employment problems of both literate and illiterate, especially women. In view of this, vermicomposting is an effective means of composting the decomposable organic wastes using earthworms naturally present in the soil. Its improves the physical and biological condition of soil, improves soil fertility and pulverizes it through their churning and turning action in addition to contributing plant nutrients, improves aeration and water holding capacity. It is the scope to small and marginal farmers in creating their own organic manure resources and ways to generate not only alternative income but maintain the soil health also. Vermicompost is a preferred nutrient source for organic farming, which enriches soil quality by improving its physicochemical and biological properties. It is ecofriendly, non-toxic, consumes low energy input for composting and is a recycled biological product. Vermicompost is becoming popular as a major component of organic farming system.

Indoor residual spraying (IRS) is the mainstay for vector control intervention of visceral leishmaniasis (VL) in India. Little is known on the control effects of IRS on different household types. Here, we assessed if IRS with insecticides has an equal residual and interventional effect on all household types in a village. We also developed a combined spatial-risk map and a sand fly, Phlebotomus argentipes density analytical model based on household characteristics, insecticide susceptibility and IRS-status to explore the spatio-temporal distributions of the vector at a micro-scale level. This study was carried out in two villages of Mahnar block in Vaishali district, Bihar. IRS using two insecticides [dichlorodiphenyltrichloroethane (DDT 50%) and synthetic pyrethroid (SP 5%)] was evaluated for VL-vector (P. argentipes) control. Temporal residual efficacy of the insecticides on different wall-surface types was evaluated using the cone-bioassay technique according to WHO guidelines. Insecticide susceptibility of local P. argentipes was explored using the tube-bioassay method. Pre- and post-IRS sand fly densities were monitored in human dwellings and animal shelters using Centers for Disease Control light-traps installed between 18:00–6:00 h. A best-fit model for sand fly density analysis was developed using multiple logistic regression analysis. Geographical information system based spatial analysis techniques were employed to map the household type distribution of insecticide susceptibility of the vector, and IRS-status of the households to interpret the spatio-temporal distributions of P. argentipes. Phlebotomus argentipes was highly susceptible to SP (100%) but showed high resistance to DDT with a 49.1% mortality rate. SP-IRS has been reported as having better community acceptance than DDT-IRS in all household types. Residual efficacies were varied between wall-surfaces; both insecticides failed to achieve the duration of IRS effectiveness recommended by the WHO. Reduction in P. argentipes counts due to SP-IRS was higher than DDT-IRS between household groups (i.e. sprayed and sentinel), in all intervals post-IRS. Combined spatial risk-maps revealed a better control effect of SP-IRS on sand flies than DDT-IRS in all household types risk-zones. The multilevel logistic regression analysis explored five risk-factors that were strongly associated with the density of P. argentipes. The results contribute to furthering current understanding of IRS-practices for control of visceral leishmaniasis in endemic Bihar, which may help in future actions for improvements.

Visceral leishmaniasis (VL) in Bihar State (India) continues to be endemic, despite the existence of effective treatment and a vector control program to control disease morbidity. A clear understanding of spatio-temporal distribution of VL may improve surveillance and control implementation. This study explored the trends in spatio-temporal dynamics of VL endemicity at a meso-scale level in Vaishali District, based on geographical information systems (GIS) tools and spatial statistical analysis.

It is general practice to utilize locally available soil for construction of geotechnical construction. This is economical way of construction. But availability of locally available waste material like fly ash, pond ash etc. gives engineers a choice to do construction in sustainable way. But for this purpose comparative study is required. In this paper comparison of geotechnical behavior of Muzaffarpur soil and locally available fly ash is done. For this purpose series of tests were conducted on the fly ash and local soil. In this study, test to find out index properties and engineering properties like; strength, compaction etc. were conducted. The potential of utilization of the fly ash as partial replacement of soil is also investigated. From the test results it is found that fly ash has better strength behavior and can be utilized as light weight construction material. It is also found that as replacement fly ash can significantly improve the strength behavior of the soil.

Essential oil extracted from different parts of the Ocimumafricanum Lour. and its chemical constituents affected by two seasons namely summer and winter growing variation were investigated. The hydro distilled essential oil content from different parts of the plant obtained from summer and winter season ranges from 0.2% to 0.6%. However, the highest essential oil content (0.3% to 0.6%) was recorded in summer and lowest oil content (0.2% to 0.3%) was recorded in winter season. The essential oil consists of Citral II is the most abundant component followed by Citral I. The highest amount of Citral I obtained from different parts of O.africanumviz., whole herb (42.5%), leaf (43.5%), stem (44.8 %) and inflorescence (39.9%) collected during summer was observed and Citral II was followed by Citral I (33.7%, 36.2%, 36.2% and 34.6% respectively). Whereas other essential oil components viz., Citronellol, Linalool and geraniol content was higher in different parts of plant obtained during winter season compared to summer season and rest of the essential oil content were higher in summer season compared to winter season. Similarly, growth parameter viz., plant height (65.8 cm), number of branches (23), number of leaves (435 no.) and leaf area (14.68 m2) were also higher in summer season compared to winter season.

Essential oil extracted from different parts of the Ocimumafricanum Lour. and its chemical constituents affected by two seasons namely summer and winter growing variation were investigated. The hydro distilled essential oil content from different parts of the plant obtained from summer and winter season ranges from 0.2% to 0.6%. However, the highest essential oil content (0.3% to 0.6%) was recorded in summer and lowest oil content (0.2% to 0.3%) was recorded in winter season. The essential oil consists of Citral II is the most abundant component followed by Citral I. The highest amount of Citral I obtained from different parts of O.africanumviz., whole herb (42.5%), leaf (43.5%), stem (44.8 %) and inflorescence (39.9%) collected during summer was observed and Citral II was followed by Citral I (33.7%, 36.2%, 36.2% and 34.6% respectively). Whereas other essential oil components viz., Citronellol, Linalool and geraniol content was higher in different parts of plant obtained during winter season compared to summer season and rest of the essential oil content were higher in summer season compared to winter season. Similarly, growth parameter viz., plant height (65.8 cm), number of branches (23), number of leaves (435 no.) and leaf area (14.68 m) were also higher in summer season compared to

Background: In vector-borne diseases such as leishmaniasis, the sand fly midgut is considered to be an important site for vector-parasite interaction. Digestive enzymes including serine peptidases such as trypsin and chymotrypsin, which are secreted in the midgut are one of the obstacles for Leishmania in establishing a successful infection. The presence of some natural inhibitors of serine peptidases (ISPs) has recently been reported in Leishmania. In the present study, we deciphered the role of these ISPs in the survival of Leishmania donovani in the hostile sand fly midgut environment.Methods: In silico and co-immunoprecipitation studies were performed to observe the interaction of L. donovani ISPs with trypsin and chymotrypsin. Zymography and in vitro enzyme assays were carried out to observe the inhibitory effect of purified recombinant ISPs of L. donovani (rLdISPs) on trypsin, chymotrypsin and the sand fly midgut peptidases. The expression of ISPs in the amastigote to promastigote transition stages were studied by semi-quantitative RT-PCR and Western blot. The role of LdISP on the survival of ISP overexpressed (OE) and ISP knocked down (KD) Leishmania parasites inside the sand fly gut was investigated by in vitro and in vivo cell viability assays.Results: We identified two ecotin-like genes in L. donovani, LdISP1 and LdISP2. In silico and co-immunoprecipitation results clearly suggest a strong interaction of LdISP molecules with trypsin and chymotrypsin. Zymography and in vitro enzyme assay confirmed the inhibitory effect of rLdISP on trypsin, chymotrypsin and the sand fly midgut peptidases. The expression of LdISP2 was found to be strongly associated with the amastigote to promastigote phase transition. The activities of the digestive enzymes were found to be significantly reduced in the infected sand flies when compared to uninfected. To our knowledge, our study is the first report showing the possible reduction of chymotrypsin activity in L. donovani infected sand flies compared to uninfected. Interestingly, during the early transition stage, substantial killing was observed in ISP2 knocked down (ISP2KD) parasites compared to wild type (WT), whereas ISP1 knocked down (ISP1KD) parasites remained viable. Therefore, our study clearly indicates that LdISP2 is a more effective inhibitor of serine peptidases than LdISP1.Conclusion: Our results suggest that the lack of ISP2 is detrimental to the parasites during the early transition from amastigotes to promastigotes. Moreover, the results of the present study demonstrated for the first time that LdISP2 has an important role in the inhibition of peptidases and promoting L. donovani survival inside the Phlebotomus argentipes midgut.

Background Indoor residual spraying (IRS) with DDT has been the primary strategy for control of the visceral leishmaniasis (VL) vector Phlebotomus argentipes in India but efficacy may be compromised by resistance. Synthetic pyrethroids are now being introduced for IRS, but with a shared target site, the para voltage-gated sodium channel (VGSC), mutations affecting both insecticide classes could provide cross-resistance and represent a threat to sustainable IRS-based disease control. Methodology/Principal findings A region of the Vgsc gene was sequenced in P. argentipes from the VL hotspot of Bihar, India. Two knockdown resistance (kdr) mutations were detected at codon 1014 (L1014F and L1014S), each common in mosquitoes, but previously unknown in phlebotomines. Both kdr mutations appear largely recessive, but as homozygotes (especially 1014F/F) or as 1014F/S heterozygotes exert a strong effect on DDT resistance, and significantly predict survivorship to class II pyrethroids in short-duration bioassays. The mutations are present at high frequency in wild P. argentipes populations from Bihar, with 1014F significantly more common in higher VL areas. Conclusions/Significance The Vgsc mutations detected appear to be a primary mechanism underlying DDT resistance in P. argentipes and a contributory factor in reduced pyrethroid susceptibility, suggesting a potential impact if P. argentipes are subjected to suboptimal levels of pyrethroid exposure, or additional resistance mechanisms evolve. The assays to detect kdr frequency changes provide a sensitive, high-throughput monitoring tool to detecting spatial and temporal variation in resistance in P. argentipes.

Extensive application of routine insecticide i.e., dichlorodiphenyltrichloroethane (DDT) to control Phlebotomus argentipes (Diptera: Psychodidae), the proven vector of visceral leishmaniasis in India, had evoked the problem of resistance/tolerance against DDT, eventually nullifying the DDT dependent strategies to control this vector. Because tolerating an hour-long exposure to DDT is not challenging enough for the resistant P. argentipes, estimating susceptibility by exposing sand flies to insecticide for just an hour becomes a trivial and futile task.Therefore, this bioassay study was carried out to investigate the maximum limit of exposure time to which DDT resistant P. argentipes can endure the effect of DDT for their survival. The mortality rate of laboratory-reared DDT resistant strain P. argentipes exposed to DDT was studied at discriminating time intervals of 60 min and it was concluded that highly resistant sand flies could withstand up to 420 min of exposure to this insecticide. Additionally, the lethal time for female P. argentipes was observed to be higher than for males suggesting that they are highly resistant to DDT's toxicity. Our results support the monitoring of tolerance limit with respect to time and hence points towards an urgent need to change the World Health Organization's protocol for susceptibility identification in resistant P. argentipes.