Phytofabrication and characterization of Alchornea cordifolia silver nanoparticles and evaluation of antiplasmodial, hemocompatibility and larvicidal potential.

Purpose: The recent emergence of Plasmodium falciparum (Pf) parasites resistant to current artemisinin-based combination therapies in Africa justifies the need to develop new strategies for successful malaria control. We synthesized, characterized and evaluated medical applications of optimized silver nanoparticles using Alchornea cordifolia (AC-AgNPs), a plant largely used in African and Asian traditional medicine. Methods: Fresh leaves of A. cordifolia were used to prepare aqueous crude extract, which was mixed with silver nitrate for AC-AgNPs synthesis and optimization. The optimized AC-AgNPs were characterized using several techniques including ultraviolet-visible spectrophotometry (UV-Vis), scanning/transmission electron microscopy (SEM/TEM), powder X-ray diffraction (PXRD), selected area electron diffraction (SAED), energy dispersive X-ray spectroscopy (EDX), Fourier transformed infrared spectroscopy (FTIR), dynamic light scattering (DLS) and Zeta potential. Thereafter, AC-AgNPs were evaluated for their hemocompatibility and antiplasmodial activity against Pf malaria strains 3D7 and RKL9. Finally, lethal activity of AC-AgNPs was assessed against mosquito larvae of Anopheles stephensi, Culex quinquefasciatus and Aedes aegypti which are vectors of neglected diseases such as dengue, filariasis and chikungunya. Results: The AC-AgNPs were mostly spheroidal, polycrystalline (84.13%), stable and polydispersed with size of 11.77 ± 5.57 nm. FTIR revealed the presence of several peaks corresponding to functional chemical groups characteristics of alkanoids, terpenoids, flavonoids, phenols, steroids, anthraquonones and saponins. The AC-AgNPs had a high antiplasmodial activity, with IC50 of 8.05 µg/mL and 10.31 µg/mL against 3D7 and RKL9 Plasmodium falciparum strains. Likewise, high larvicidal activity of AC-AgNPs was found after 24 h- and 48 h-exposure: LC50 = 18.41 µg/mL and 8.97 µg/mL (Culex quinquefasciatus), LC50 = 16.71 µg/mL and 7.52 µg/mL (Aedes aegypti) and LC50 = 10.67 µg/mL and 5.85 µg/mL (Anopheles stephensi). The AC-AgNPs were highly hemocompatible (HC50 > 500 µg/mL). Conclusion: In worrying context of resistance of parasite and mosquitoes, green nanotechnologies using plants could be a cutting-edge alternative for drug/insecticide discovery and development.

Laboratory evaluation of a new alphacypermethrin long-lasting insecticidal net against Anopheles culicifacies s.l.

Long-lasting insecticidal nets (LLINs) are being promoted by World Health Organization (WHO) as key vector control tool for the prevention and control of malaria and other vector-borne diseases. A laboratory study was conducted to evaluate the bio-efficacy of a new alphacypermethrin LN (HILNET®) against insecticide-susceptible Anopheles culicifacies s.l.. This study presents the observations on (a) regeneration time, the period required to regenerate insecticide in the LLIN after three consecutive WHO standard washes in a single day to deplete the insecticide on the net surface, and (b) wash resistance of the LLIN to retain bio-efficacy up to 20 repeated washes at intervals corresponding to the regeneration time in days. In the present evaluation, the regeneration time of HILNet® was determined as 2 days. Wash resistance studies of HILNet® showed a knockdown rate of 100% up to 15 washes and 97% at the 20th wash and complied with the criteria of WHO knockdown rate of ≥ 95%. The mean active ingredient content from 0 to 20 washes was in the range of 5.8 to 6.04 g/kg and met the WHO criteria of the product claim (± 25% g/kg). The insecticide un-impregnated control net has shown no detectable levels of alphacypermethrin. In this evaluation, HILNet® fulfilled the WHO laboratory (phase I) efficacy criteria based on the rates of knockdown up to 20 standard WHO washes.

Small-scale field evaluation of the entomological efficacy and the residual activity of Fludora® Fusion WP-SB indoor residual spraying against Anopheles culicifacies s.l. in Gujarat, India.

OBJECTIVES: To evaluate the entomological efficacy and the residual activity of indoor residual spraying with Fludora® Fusion 562.5 WP-SB, a combination formulation containing clothianidin, a neonicotinoid and deltamethrin, a pyrethroid, against the main rural malaria vector, Anopheles culicifacies s.l., in India in a small-scale trial. METHODS: In three study villages, suitable households were randomly allocated to five treatments: Fludora® Fusion 562.5 WP-SB (target dose 225 mg active ingredient AI/m2 ); clothianidin 70 WG (target dose 200 mg AI/m2 ); K-Othrine 250 WG (deltamethrin, target dose 25 mg AI/m2 ); Ficam VC 80 WP-SB (bendiocarb, target dose 400 mg AI/m2 ) and unsprayed control. Insecticides were sprayed by hand compression sprayers with control flow valves and 8002E nozzles. Post-spray cone bioassays were done on insecticide-treated walls using a colonised, deltamethrin-resistant strain of An. culicifacies. Mosquitoes were collected from treated rooms by different methods. The insecticide content on filter papers collected from the sprayed walls was determined by chemical assay to assess the spray quality. RESULTS: The ratios of applied to target doses of insecticides were within 0.84 to 1.4, showing a good spray quality. The cone bioassays revealed residual action lasting 7 months for all insecticides without significant differences in mortality between different surfaces treated nor between the four treatment arms (P > 0.05). Considering all entomological parameters such as indoor resting density, excito-repellency, blood-feeding inhibition and delayed mortality, the overall efficacy of Fludora® Fusion WG-SB was equal or better compared with other insecticides. CONCLUSIONS: Fludora® Fusion showed overall equal or better efficacy than deltamethrin and bendiocarb alone against a pyrethroid-resistant malaria vector population and can be considered as an alternative product for management of pyrethroid resistance in malaria vectors.

Characterization and monitoring of deltamethrin-resistance in Anopheles culicifacies in the presence of a long-lasting insecticide-treated net intervention.

BACKGROUND: Deltamethrin-impregnated, long-lasting insecticidal nets (LLINs) were distributed in the study area from November 2014 to January 2015 to evaluate their impact on malaria transmission in the presence of insecticide-resistant vectors. Studies were carried out in 16 selected clusters in Keshkal sub-district, Chhattisgarh State, India to monitor and characterize deltamethrin resistance in Anopheles culicifacies sensu lato. RESULTS: Deltamethrin susceptibility of An. culicifacies decreased in a post-LLIN survey compared to a pre-LLIN survey and was not significant (p > 0.05) while, the knockdown values showed significant increase (p < 0.05). Pre-exposure to piperonyl butoxide, triphenyl phosphate showed synergism against deltamethrin (p < 0.001). Biochemical assays showed significantly (p < 0.05) elevated monooxygenases in 3 of 5 clusters in post-LLIN survey-I that increased to 10 of 11 clusters in post-LLIN survey-II, while esterases were found significantly elevated in all clusters and both enzymes were involved in conferring pyrethroid resistance, not discounting the involvement of kdr (L1014L/S) gene that was heterozygous and at low frequency (4-5%). CONCLUSION: This field study, in a tribal district of India, after distribution of deltamethrin-impregnated LLINs showed decrease in deltamethrin susceptibility in An. culicifacies, a major vector of malaria in this study area and in India. Results indicated development of resistance as imminent with the increase in insecticide selection pressure. There is an urgent need to develop new vector control tools, with insecticide classes having novel mechanisms of resistance, to avoid or delay the onset of resistance. Regular insecticide resistance monitoring and mechanistic studies should be the priority for the malaria control programmes to suggest strategies for insecticide resistance management. The global commitment to eliminate malaria by 2030 needs various efforts that include development of combination vector control products and interventions and few are becoming available.

Field testing & evaluation of the efficacy & duration of effectiveness of a biolarvicide, Bactivec® SC (Bacillus thuringiensis var. israelensis SH-14) in Bengaluru, India.

Background & objectives: Different formulations of Bacillus thuringiensis var. israelensis (Bti) have been tested against different mosquito vectors and other insects for their residual activity. In the present study, the efficacy and residual activity of a new formulation of Bti (Bactivec Suspension Concentrate) were evaluated against immature stages of Anopheles stephensi Liston (Diptera: Culicidae), Aedes aegypti Linnaeus (Diptera: Culicidae) and Culex quinquefasciatus Say (Diptera: Culicidae), in natural habitats in Phase II and Phase III in Bengaluru, India. Methods: Preferential breeding habitats of the mosquito species were selected and four dosages (0.25, 0.5, 1 and 2 ml/50 l) were tested in Phase II trial. Two most effective dosages, 0.5 and 1 ml/50 l were selected for Phase III trial. The evaluation was carried out essentially following the guidelines of the World Health Organization Pesticide Evaluation Scheme. Pre-treatment and post-treatment densities were recorded at regular intervals, and >80 per cent reduction in pupae was taken as the duration of effectiveness. Results: Bactivec SC treated at the dosage of 1 ml/50 l could produce 10-17 days efficacy (>80% reduction in pupae) in clean water habitats tested, whereas 0.5 ml/50 l dosage showed residual activity from 7 to 14 days against Ae. aegypti and An. stephensi in Phase III studies. In polluted water habitats, 4-7 days efficacy could be recorded against Cx. quinquefasciatus in Phase III. Interpretation & conclusions: The Bactivec SC formulation was operationally feasible and easy to handle. For the control of Anopheles and Aedes mosquitoes in freshwater habitats, 1 ml/50 l dosage was found effective, whereas in polluted water habitats against Cx. quinquefasciatus 5 ml/m2 was found effective.

Impact of long-lasting insecticidal nets on prevalence of subclinical malaria among children in the presence of pyrethroid resistance in Anopheles culicifacies in Central India.

BACKGROUND: Subclinical (asymptomatic) cases of malaria could be a major barrier to the success of malaria elimination programs. This study has evaluated the impact of long-lasting insecticidal nets (LLINs) on the prevalence of subclinical malaria in the presence of pyrethroid resistance in the main malaria vector Anopheles culicifacies on malaria transmission among a cohort of children in villages of the Keshkal sub-district in Chhattisgarh state. METHODS: A cohort of 6582 children ages less than 14 years was enrolled from 80 study clusters. Post monsoon survey was carried out at baseline before LLIN distribution, and 5862 children were followed up in the subsequent year. Study outcomes included assessment of subclinical malarial infections and use of LLINs among the study cohort in the presence of varied levels of pyrethroid resistance. FINDINGS: In the baseline survey, the proportion of subclinical malaria was 6·1%. LLIN use during the previous night was 94·8%. Overall, prevalence of subclinical malaria was significantly reduced to 1% (p<0·001) in the second survey. LLIN users were protected from malaria (OR: 0·25, 95% CI=0·12-0·52, p<0.001) and subclinical malaria (OR: 0·25, 95% CI=0·11-0·58, p=0·001) despite the presence of pyrethroid resistance in the study area. INTERPRETATION: In this low transmission area, sleeping under LLINs significantly reduced the burden of malaria among children. In the presence of pyrethroid resistant malaria vector, a high LLIN use of 94·5% was observed to have significantly brought down the proportion of subclinical malaria among the cohort children.

Small-scale evaluation of the efficacy and residual activity of alpha-cypermethrin WG (250 g AI/kg) for indoor spraying in comparison with alpha-cypermethrin WP (50 g AI/kg) in India.

BACKGROUND: Indoor residual spraying (IRS) with different formulations of insecticides is being used for the control of mosquito vectors in many countries. In the present study, residual efficacy and duration of effectiveness of IRS with alpha-cypermethrin WG-SB (250 g AI/m2) formulation was compared with WP formulation (50 g AI/kg) in a small scale (Phase II) field trial. METHODS: Two dosages, i.e. 20 and 30 mg AI/m2, were used and the efficacy and duration of effectiveness was assessed on alpha-cypermethrin susceptible population of Anopheles stephensi. Four types of surfaces were selected, namely cement wall with distemper coating, cement wall with lime coating, mud wall with lime coating, and brick wall unpainted. Spraying was carried out with Hudson sprayer fitted with control flow valve. Bioassays were carried out at weekly and then fortnightly intervals. Chemical analysis of filter paper samples collected from the sprayed surfaces was done at Walloon Agricultural Research Institute, Gembloux, Belgium. RESULTS: Alpha-cypermethrin WG-SB showed residual efficacy (>80% mortality) for 13-15 weeks for the 20 mg/m2 dosage and 13-16 weeks for the 30 mg/m2 dosage, whereas alpha-cypermethrin WP showed residual efficacy for 11-15 weeks for the 20 mg/m2 dosage and 11-14 weeks for the 30 mg/m2 dosage on the surfaces tested. The average of the applied to target dose ratio ranged from 0.89 to 1.17 for alpha-cypermethrin WG-SB at 20 mg AI/m2, 0.83-1.80 for the WG-SB at 30 mg AI/m2, 0.87-1.66 for alpha-cypermethrin WP at 20 mg AI/m2, and 0.68-1.06 for WP at 30 mg AI/m2. No adverse events were reported, either by the spray men or the household inhabitants, during and after the spray operations. CONCLUSIONS: The results suggest that the dose of WG 30 mg/m2 gave slightly longer effective residual action against malaria vector (16 weeks) on most common indoor surfaces and could be used for effective control of Anopheles mosquitoes. The WG formulation was found to be easy to handle, no smell was reported during the spraying and was found to be operationally acceptable for indoor residual spraying.

Adulticidal & larvicidal efficacy of three neonicotinoids against insecticide susceptible & resistant mosquito strains.

BACKGROUND & OBJECTIVES: Due to ever growing insecticide resistance in mosquitoes to commonly used insecticides in many parts of the globe, there is always a need for introduction of new insecticides for the control of resistant vector mosquitoes. In this study, larvicidal and adulticidal efficacies of three neonicotinoids (imidacloprid, thiacloprid and thiamethoxam) were tested against resistant and susceptible populations of Anopheles stephensi Liston 1901, Aedes (Stegomyia) aegypti Linnaeus, and Culex quinquefasciatus Say (Diptera: Culicidae). METHODS: Laboratory-reared mosquito species were used. Insecticide susceptibility tests were done using standard WHO procedures and using diagnostic dosages of insecticide test papers and larvicides. Adulticidal efficacy of candidate insecticides was assessed using topical application method and larval bioassays were conducted using standard WHO procedure. RESULTS: The results of topical application on 3-5 day old female mosquitoes indicated that resistant strain of An. stephensi registered lower LC 50 values than the susceptible strain. Among the three insecticides tested, thiacloprid was found more effective than the other two insecticides. Culex quinquefasciatus registered lowest LC 50 for imidacloprid than the other two mosquito species tested. In larval bioassays, the LC 50 values registered for imidacloprid were in the order of Cx. quinquefasciatus < An. stephensi (SS) < An. stephensi (RR) < Ae. aegypti. In case of thiacloprid, the order of efficacy (LC 50 ) was Cx. quinquefasciatus < An. stephensi (SS) < An. stephensi (RR), whereas in case of thiamethoxam, the larvicidal efficacy was in the order of An. stephensi (RR) < An. stephensi (SS) < Cx. quinquefasciatus. INTERPRETATION & CONCLUSIONS: The present study indicated that insecticide resistant strains of mosquito species tested showed more susceptibility to the three neonicotinoids tested, and the possibility of using neonicotinoids for the control of resistant mosquitoes should be explored.

An oxidative amidation and heterocyclization approach for the synthesis of ß-carbolines and dihydroeudistomin Y.

A novel synthetic methodology has been developed for the synthesis of dihydro-ß-carboline derivatives employing oxidative amidation-Bischler-Napieralski reaction conditions using tryptamine and 2,2-dibromo-1-phenylethanone as key starting materials. A number of dihydro-ß-carboline derivatives have been synthesized in moderate to good yields using this methodology. Attempts were made towards the conversion of these dihydro-ß-carbolines to naturally occurring eudistomin alkaloids.

Effectiveness and durability of Interceptor® long-lasting insecticidal nets in a malaria endemic area of central India.

BACKGROUND: In the present study, Interceptor®, long-lasting polyester net, 75 denier and bursting strength of minimum 250 kPa coated with alpha-cypermethrin @ 200 mg/m² was evaluated for its efficacy in reducing the mosquito density, blood feeding inhibition and malaria incidence in a tribal dominated malaria endemic area in Chhattisgarh state, central India. Its durability, washing practices and usage pattern by the community was also assessed up to a period of three years. METHODS: The study was carried out in two phases. In the first phase (September 2006 to August 2007), 16 malaria endemic villages in district Kanker were randomized into three groups, viz. Interceptor net (LN), untreated polyester net (100 denier) and without net. Malaria cases were detected by undertaking fortnightly surveillance by home visits and treated as per the national drug policy. Mosquito collections were made by hand catch and pyrethrum space spray methods from human dwellings once every month. Slide positivity rate (SPR) and malaria incidence per 1000 population (PI) were compared between the three study arms to assess the impact of use of Interceptor nets. Simultaneously, wash resistance studies were carried out in the laboratory by doing cone bioassays on Interceptor LNs washed up to 20 times. Activities undertaken in second Phase (April 2008 to October 2009) after an interval of about 18 months post-net distribution included questionnaire based surveys at every six months, i.e. 18, 24, 30 and 36 months to observe durability, usage pattern of LNs and washing practices by the community. After 36 months of field use, 30 nets were retrieved and sampled destructively for chemical analysis. RESULTS: Interceptor nets were found effective in reducing the density, parity rate and blood feeding success rate of main malaria vector Anopheles culicifacies as compared to that in untreated net and no net villages. SPR in LN villages was 3.7% as compared to 6.5% in untreated and 11% in no net villages. PI in LN villages was 16.4 in comparison to 24.8 and 44.2 in untreated polyester net and no net villages respectively. In surveys carried out after three years of initial distribution, 78.7% (737/936) nets were still in possession with the households, of which 68% were used every night. An. culicifacies mortality was >80% in cone bioassays done on LNs washed up to 20 times in laboratory. Mean alpha-cypermethrin content was 43.5 ± 31.7 mg/m² on Interceptor LNs withdrawn after three years of household use against the baseline specification of 200 mg/m². A gradual increase in the proportion of holed nets was observed with the increased period of usage. CONCLUSION: Interceptor nets were highly effective in reducing vector densities as well as malaria incidence in the study villages. Availability of 78% nets with the households in usable condition clearly indicated durability of Interceptor LNs up to three years in the rural setting of India. The nets were found to contain an effective concentration of alpha-cypermethrin against malaria vector after three years of household use.

Silica nanoparticle: a potential new insecticide for mosquito vector control.

Presently, there is a need for increased efforts to develop newer and effective methods to control mosquito vectors as the existing chemical and biological methods are not as effective as in earlier period owing to different technical and operational reasons. The use of nanomaterial products in various sectors of science including health increased during the last decade. We tested three types of nanosilica, namely lipophilic, hydrophilic and hydrophobic, to assess their larvicidal, pupicidal and growth inhibitor properties and also their influence on oviposition behaviour (attraction/deterrence) of mosquito species that transmit human diseases, namely malaria (Anopheles), yellow fever, chickungunya and dengue (Aedes), lymphatic filariasis and encephalitis (Culex and Aedes). Application of hydrophobic nanosilica at 112.5 ppm was found effective against mosquito species tested. The larvicidal effect of hydrophobic nanosilica on mosquito species tested was in the order of Anopheles stephensi > Aedes aegypti > Culex quinquefasciatus, and the pupicidal effect was in the order of A. stephensi > C. quinquefasciatus > Ae. aegypti. Results of combined treatment of hydrophobic nanosilica with temephos in larvicidal test indicated independent toxic action without any additive effect. This is probably the first report that demonstrated that nanoparticles particularly nanosilica could be used in mosquito vector control.

Salivary gland transcriptome analysis during Plasmodium infection in malaria vector Anopheles stephensi.

BACKGROUND: Understanding the tissue-specific molecular cross-talk mechanism during the mosquito-parasite interaction is of prime importance in the design of new strategies for malaria control. Because mosquito salivary glands are the final destination for the parasite maturation and transmission of vector-borne diseases, identification and characterization of salivary genes and their products are equally important in order to access their effect on the infectivity of the parasite. During the last five years there have been several studies on the sialomes of Anopheles mosquitoes, however very limited information is available on the changes in the salivary gland transcriptome in the presence of Plasmodium, and this information is limited to the mosquito Anopheles gambiae. METHODS: In this study we aimed to explore and identify parasite-induced transcripts from the salivary glands of Anopheles stephensi, using a subtractive hybridization protocol. RESULTS: Ninety-four percent of expressed sequence tags (ESTs) showed close homology to previously known families of mosquito salivary gland secretary proteins, representing the induced expression of alternative splicing and/or additional new members of the protein family. The remaining 6% of ESTs did not yield significant homology to any known proteins in the non-redundant database and thus may represent a class of unknown/novel salivary proteins. Primary analysis of the ESTs also revealed identification of several novel immune-related transcripts, including defensin and cecropins, probably involved in counter-activation of the antagonistic defense system. A comprehensive description of each family of proteins has been discussed in relation to the tissue-specific mosquito-parasite interaction. CONCLUSION: This is the first report on the identification of new putative salivary genes, presumably activated during parasite infection.