Cone Bioassays Research Articles

Evaluation of Long-Lasting Insecticidal Nets (DuraNet®) Under laboratory and Semi-Field Conditions Using Experimental Huts Against Anopheles Mosquitoes in Jimma Zone, Southwestern Ethiopia

Background:Long-Lasting Insecticidal Nets (LLINs) efficacy could be compromised due to a lot of influences together with user compliance and vector population insecticide resistance status. Thus, this study was to assess the biological efficacy of DuraNet® with the help of the World Health Organization cone bioassay and field experimental hut.Methods:A laboratory and a semi-field conditions experimental huts against Anopheles Mosquitoes were conducted in southwestern Ethiopia from September 2015 to January 2016. The bio efficacy of DuraNet® was evaluated using the WHO cone bioassay test and then its field efficacy was evaluated using experimental huts against the malaria vector population.Results:World Health Organization cone bioassay tests against pyrethroid-resistant An. arabiensis led to mean percent mortality and knockdown of 78% and 93%, respectively. Washing of DuraNet® successively reduced its efficacy from 93% knockdown (0 wash) to 45% knockdown (20 washes). Similarly, mean mortality decreased from 84% (0 wash) to 47% (20 washes). A total of 1575 female mosquitoes were collected over 40 nights out of which 1373(87.8%) were An. gambiae s.l., 116 (7.4%) were Anopheles coustani and 107 (6.8%) were An. pharoensis. The mean blood-feeding rate was significantly lower (P < .001) in hut containing unwashed DuraNet® when compared to hut containing untreated DuraNet®. The mean mortality rate was significantly higher (P < .001) in hut containing DuraNet® when compared to hut containing untreated DuraNet®. Unwashed DuraNet® showed the highest personal protection 88.7% and 100% against An. Arabiensis and An. pharoensis, respectively.Conclusion:Both DuraNet® and PermaNet 2.0 moderate efficacy against a pyrethroid-resistant population of An. arabiensis from Ethiopia. The bio efficacy of DuraNet® was found below the WHO recommendation. Therefore, the real impact of the observed insecticide resistance against DuraNet® to be further studied under phase-III trials, the need for new alternative vector control tools remains critical.

Increased prevalence of insecticide resistance in Anopheles coluzzii populations in the city of Yaoundé, Cameroon and influence on pyrethroid-only treated bed net efficacy.

In Cameroon, pyrethroid-only long-lasting insecticidal nets (LLINs) are still largely used for malaria control. The present study assessed the efficacy of such LLINs against a multiple-resistant population of the major malaria vector, Anopheles coluzzii, in the city of Yaoundé via a cone bioassay and release-recapture experimental hut trial. Susceptibility of field mosquitoes in Yaoundé to pyrethroids, DDT, carbamates and organophosphate insecticides was investigated using World Health Organization (WHO) bioassay tube tests. Mechanisms of insecticide resistance were characterised molecularly. Efficacy of unwashed PermaNet® 2.0 was evaluated against untreated control nets using a resistant colonised strain of An. coluzzii. Mortality, exophily and blood feeding inhibition were estimated. Field collected An. coluzzii displayed high resistance with mortality rates of 3.5% for propoxur (0.1%), 4.16% for DDT (4%), 26.9% for permethrin (0.75%), 50.8% for deltamethrin (0.05%), and 80% for bendiocarb (0.1%). High frequency of the 1014F west-Africa kdr allele was recorded in addition to the overexpression of several detoxification genes, such as Cyp6P3, Cyp6M2, Cyp9K1, Cyp6P4 Cyp6Z1 and GSTe2. A low mortality rate (23.2%) and high blood feeding inhibition rate (65%) were observed when resistant An. coluzzii were exposed to unwashed PermaNet® 2.0 net compared to control untreated net (p < 0.001). Furthermore, low personal protection (52.4%) was observed with the resistant strain, indicating reduction of efficacy. The study highlights the loss of efficacy of pyrethroid-only nets against mosquitoes exhibiting high insecticide resistance and suggests a switch to new generation bed nets to improve control of malaria vector populations in Yaoundé.

First detection of voltage-gated sodium channel mutations in Phlebotomus argentipes collected from Bangladesh

Phlebotomus argentipes is the main vector of visceral leishmaniasis in Bangladesh and is controlled using deltamethrin, a synthetic pyrethroid, through indoor residual spraying (IRS). A mutation at L1014 (leucine at codon 1014) of the voltage-gated sodium channel (VGSC), known as a knockdown resistance (kdr) gene, is thought to be an important pyrethroid resistance mechanism. This study detected mutations at codon 1014, and at codons 1011, 1016, and 1020, which are kdr sites in other insects. The kdr relationship with deltamethrin resistance in P. argentipes from an IRS-targeted site in Bangladesh was also evaluated. Sand flies were collected from Magurjora village, Mymensingh district, Bangladesh in November 2012. A WHO cone bioassay test using deltamethrin was conducted and specimens were grouped as 'live' or 'dead'. After morphological identification, genomic DNA was used to genotype a partial VGSC gene from P. argentipes. The kdr/ pyrethroid resistance relationship was evaluated using Fisher's exact test. Targeted codons were genotyped from 8 'live' and 63 'dead' P. argentipes. All 'live' specimens had mutant alleles (L1014F and L1014S) at codon 1014. The mutant allele rate was 94% for 'live' specimens and 55% for 'dead' specimens. The mutant allele survival odds were higher for the wild-type L1014L allele, and L1014F odds were lower for L1014S. There were no mutations at codons 1011, 1016, and 1020. The L1014 mutations suggested that pyrethroid resistance had appeared in Bangladesh. Further research on kdr mutations in P. argentipes is important for the appropriate IRS.

A study of malaria vector surveillance as part of the Malaria Elimination Demonstration Project in Mandla, Madhya Pradesh

BackgroundUnderstanding of malaria vector density, distribution, insecticide resistance, vector incrimination, infection status, and identification of sibling species are some of the essential components of vector control measures for achieving malaria elimination goals.MethodsAs part of the malaria elimination demonstration project, entomological surveillance was carried out from October 2017 to October 2019 by collecting indoor resting mosquitoes using hand catch method. Susceptibility test was done for determining the insecticide resistance status of vector mosquito Anopheles culicifacies using standard protocols by the World Health Organization. The cone bioassay method was used for determining the efficacy and quality of insecticide sprayed. Mosquitoes collected from different ecotypes were identified and processed for parasite identification, vector incrimination and sibling species determination.ResultsThe two known malaria vector species (Anopheles culicifacies and Anopheles fluviatilis) were found in the study area, which have been previously reported in this and adjoining areas of the State of Madhya Pradesh. The prevalence of An. culicifacies was significantly higher in all study villages with peak in July while lowest number was recorded in May. Proportion of vector density was observed to be low in foothill terrains. The other anopheline species viz, Anopheles subpictus, Anopheles annularis, Anopheles vagus, Anopheles splendidus, Anopheles pallidus, Anopheles nigerrimus and Anopheles barbirostris were also recorded in the study area, although their prevalence was significantly less compared to the An. culicifacies. In 2017, An. culicifacies was found to be resistant to dichloro-diphenyl-trichloroethane (DDT) and malathion, with possible resistance to alphacypermethrin and susceptible to deltamethrin. However, in 2019, the species was found to be resistant to alphacypermethrin, DDT, malathion, with possible resistance to deltamethrin. The bioassays revealed 82 to > 98% corrected % mortality of An. culicifacies on day-one post-spraying and 35 to 62% on follow-up day-30. Anopheles culicifacies sibling species C was most prevalent (38.5%) followed by A/D and E while B was least pre-dominant (11.9%). Anopheles fluviatilis sibling species T was most prevalent (74.6%) followed by U (25.4%) while species S was not recorded. One An.culicifacies (sibling species C) was found positive for Plasmodium falciparum by PCR tests in the mosquitoes sampled from the test areas.ConclusionBased on the nine entomologic investigations conducted between 2017–2019, it was concluded that An. culicifacies was present throughout the year while An. fluviatilis had seasonal presence in the study areas. Anopheles culicifacies was resistant to alphacypermethrin and emerging resistance to deltamethrin was observed in this area. Anopheles culicifacies was confirmed as the malaria vector. This type of information on indigenous malaria vectors and insecticide resistance is important in implementation of vector control through indoor residual spraying (IRS) and use of insecticide-impregnated bed nets for achieving the malaria elimination goals.

Efficacy evaluation of Veeralin LN, a PBO-incorporated alpha-cypermethrin long-lasting insecticidal net against Anopheles culicifacies in experimental huts in Odisha State

BackgroundThe success of malaria control using long-lasting insecticidal nets (LLINs) is threatened by pyrethroid resistance developed by the malaria vectors, worldwide. To combat the resistance, synergist piperonyl butoxide (PBO) incorporated LLINs is one of the available options. In the current phase II hut trial, the efficacy of Veeralin®LN (an alpha-cypermethrin and PBO-incorporated net) was evaluated against Anopheles culicifacies, a pyrethroid resistant malaria vector.MethodsThe performance of Veeralin®LN was compared with MAGNet®LN and untreated net in reducing the entry, induced exit, mortality and blood feeding inhibition of target vector species.ResultsThe performance of Veeralin was equal to MAGNet in terms of reducing hut entry, inhibiting blood feeding and inducing exophily, and with regard to causing mortality Veeralin was better than MAGNet. When compared to untreated net, a significant reduction in hut entry and blood feeding and an increase in exophily and mortality were observed with Veeralin. In cone bioassays, unwashed Veeralin caused > 80% mortality of An. culicifacies.ConclusionsVeeralin performed equal to (entry, exit, feeding) or better than (mortality in huts and cone bioassays) MAGNet and could be an effective tool against pyrethroid resistant malaria vectors.

Short Persistence and Vector Susceptibility to Ficam 80WP (bendiocarb active ingredient) During Pilot Application of Indoor Residual Spraying in Burkina Faso, West Africa.

Indoor residual spraying (IRS) was applied in addition to the use of long-lasting insecticidal nets in the South West in Burkina Faso, where Anopheles gambiae s.l. the major malaria vector was resistant to pyrethroids. This study was designed to evaluate the efficacy and residual life of bendiocarb (active ingredient) used for spraying on different wall surfaces (mud and cement). Cone bioassays were done monthly with the susceptible An. gambiae 'Kisumu' strain and the local wild populations to determine the duration for which insecticide was effective in killing mosquitoes. Cone bioassay data showed low efficacy and short persistence of bendiocarb applied on mud and cement walls, lasting 2 mo with the susceptible insectary strain and less than 1 mo with An. gambiae wild populations. In addition, WHO tube assays confirmed resistance of An. gambiae wild populations to 0.1% bendiocarb with mortality rates less than 80% in both study sites (sprayed and unsprayed sites). The pilot study of IRS with bendiocarb showed that the residual efficacy of bendiocarb was very short, and resistance to bendiocarb was confirmed in wild populations of An. gambiae s.l. Therefore, Ficam 80 WP was not suitable for IRS in this area due to the short residual duration related mainly to vectors resistance to bendiocarb. While waiting for innovative malaria control tool, alternative insecticide (organophosphate or neonicotinoid classes) or combinations of insecticides have to be used for insecticide resistance management in Burkina Faso.

Malaria vector species composition and entomological indices following indoor residual spraying in regions bordering Lake Victoria, Tanzania

BackgroundVector control through long-lasting insecticidal nets (LLINs) and focal indoor residual spraying (IRS) is a major component of the Tanzania national malaria control strategy. In mainland Tanzania, IRS has been conducted annually around Lake Victoria basin since 2007. Due to pyrethroid resistance in malaria vectors, use of pyrethroids for IRS was phased out and from 2014 to 2017 pirimiphos-methyl (Actellic® 300CS) was sprayed in regions of Kagera, Geita, Mwanza, and Mara. Entomological surveillance was conducted in 10 sprayed and 4 unsprayed sites to determine the impact of IRS on entomological indices related to malaria transmission risk.MethodsWHO cone bioassays were conducted monthly on interior house walls to determine residual efficacy of pirimiphos-methyl CS. Indoor CDC light traps with or without bottle rotator were hung next to protected sleepers indoors and also set outdoors (unbaited) as a proxy measure for indoor and outdoor biting rate and time of biting. Prokopack aspirators were used indoors to capture resting malaria vectors. A sub-sample of Anopheles was tested by PCR to determine species identity and ELISA for sporozoite rate.ResultsAnnual IRS with Actellic® 300CS from 2015 to 2017 was effective on sprayed walls for a mean of 7 months in cone bioassay. PCR of 2016 and 2017 samples showed vector populations were predominantly Anopheles arabiensis (58.1%, n = 4,403 IRS sites, 58%, n = 2,441 unsprayed sites). There was a greater proportion of Anopheles funestus sensu stricto in unsprayed sites (20.4%, n = 858) than in sprayed sites (7.9%, n = 595) and fewer Anopheles parensis (2%, n = 85 unsprayed, 7.8%, n = 591 sprayed). Biting peaks of Anopheles gambiae sensu lato (s.l.) followed periods of rainfall occurring between October and April, but were generally lower in sprayed sites than unsprayed. In most sprayed sites, An. gambiae s.l. indoor densities increased between January and February, i.e., 10–12 months after IRS. The predominant species An. arabiensis had a sporozoite rate in 2017 of 2.0% (95% CI 1.4–2.9) in unsprayed sites compared to 0.8% (95% CI 0.5–1.3) in sprayed sites (p = 0.003). Sporozoite rates were also lower for An. funestus collected in sprayed sites.ConclusionThis study contributes to the understanding of malaria vector species composition, behaviour and transmission risk following IRS around Lake Victoria and can be used to guide malaria vector control strategies in Tanzania.

Fine scale spatial investigation of multiple insecticide resistance and underlying target-site and metabolic mechanisms in Anopheles gambiae in central C\xf4te d\u2019Ivoire

Routine monitoring of occurrence, levels and mechanisms of insecticide resistance informs effective management strategies, and should be used to assess the effect of new tools on resistance. As part of a cluster randomised controlled trial evaluating a novel insecticide-based intervention in central Côte d’Ivoire, we assessed resistance and its underlying mechanisms in Anopheles gambiae populations from a subset of trial villages. Resistance to multiple insecticides in An. gambiae s.s. and An. coluzzii was detected across villages, with dose–response assays demonstrating extremely high resistance intensity to the pyrethroid deltamethrin (> 1,500-fold), and mortality following exposure to pyrethroid-treated bednets was low (< 30% mortality in cone bioassays). The 1014F kdr mutation was almost fixed (≥ 90%) in all villages but the 1575Y kdr-amplifying mutation was relatively rare (< 15%). The carbamate and organophosphate resistance-associated Ace-1 G119S mutation was also detected at moderate frequencies (22–43%). Transcriptome analysis identified overexpression of P450 genes known to confer pyrethroid resistance (Cyp9K1, Cyp6P3, and Cyp6M2), and also a carboxylesterase (COEAE1F) as major candidates. Cyp6P3 expression was high but variable (up to 33-fold) and correlated positively with deltamethrin resistance intensity across villages (r2 = 0.78, P = 0.02). Tools and strategies to mitigate the extreme and multiple resistance provided by these mechanisms are required in this area to avoid future control failures.

Efficacy of Fludora\xae Fusion (a mixture of deltamethrin and clothianidin) for indoor residual spraying against pyrethroid-resistant malaria vectors: laboratory and experimental hut evaluation

BackgroundA new generation of IRS insecticides which can provide improved and prolonged control of pyrethroid-resistant malaria vector populations are being developed. Fludora® Fusion is a new IRS insecticide containing a mixture of deltamethrin and clothianidin, a neonicotinoid.MethodsThe efficacy of Fludora® Fusion IRS was evaluated over 11–12 months on concrete and mud substrates in laboratory bioassays and experimental huts against wild free-flying pyrethroid-resistant Anopheles gambiae (sensu lato) in Cové, Benin. A comparison was made with the two active ingredients of the mixture; clothianidin and deltamethrin, applied alone. CDC bottle bioassays were also performed to investigate resistance to clothianidin in the wild vector population.ResultsFludora® Fusion induced > 80% laboratory cone bioassay mortality with both susceptible and pyrethroid-resistant An. gambiae (s.l.) for 7–9 months on concrete block substrates and 12 months on mud block substrates. The vector population at the experimental hut site was fully susceptible to clothianidin in CDC bottle bioassays. Overall mortality rates of wild free-flying pyrethroid-resistant An. gambiae (s.l.) entering the experimental huts during the 11-month trial were < 15% with deltamethrin and significantly higher with Fludora® Fusion (69–71%) and clothianidin alone (72–78%). Initial high experimental hut mortality rates with Fludora® Fusion (> 80%) only declined by 50% after 8 months. Monthly in situ wall cone bioassay mortality of susceptible mosquitoes was > 80% for 9–12 months with Fludora® Fusion and clothianidin alone. Fludora® Fusion induced significantly higher levels of early exiting of mosquitoes compared to clothianidin alone (55–60% vs 37–38%, P < 0.05).ConclusionsIndoor residual spraying with Fludora® Fusion induced high and prolonged mortality of wild pyrethroid-resistant malaria vectors for 7–10 months mostly due to the clothianidin component and substantial early exiting of mosquitoes from treated huts due to the pyrethroid component. Fludora® Fusion is an important addition to the current portfolio of IRS insecticides with the potential to significantly reduce transmission of malaria by pyrethroid-resistant mosquito vectors.

Indoor spraying with chlorfenapyr (a pyrrole insecticide) provides residual control of pyrethroid-resistant malaria vectors in southern Benin

BackgroundNew classes of insecticides with novel modes of action, which can provide effective and prolonged control of insecticide-resistant malaria vector populations, are urgently needed for indoor residual spraying. Such insecticides can be included in a rotation plan to manage and prevent further development of resistance in mosquito vectors of malaria. Chlorfenapyr, a novel pyrrole insecticide with a unique mode of action, is being developed as a long-lasting IRS formulation.MethodsThe efficacy of several formulations of chlorfenapyr alone and as mixtures with alpha-cypermethrin were evaluated in an experimental hut trial against wild pyrethroid-resistant Anopheles gambiae sensu lato in Cové, Benin, in an attempt to identify the most effective and long-lasting formulations for IRS. The trial lasted 12 months. A comparison was made with alpha-cypermethrin and bendiocarb formulations. CDC bottle bioassays were performed to investigate cross-resistance to chlorfenapyr in the local vector population.ResultsMortality rates in World Health Organization (WHO) cylinder bioassays were < 5% with pyrethroids due to high levels of pyrethroid resistance, but > 95% with bendiocarb thus confirming susceptibility to carbamates in the vector population. CDC bottle bioassays showed no cross-resistance between pyrethroids and chlorfenapyr. Overall mortality of free-flying mosquitoes entering the experimental huts over the 12-month trial was 4% with alpha-cypermethrin and 12% with bendiocarb. The chlorfenapyr solo-formulations induced significantly higher levels of mortality (38–46%) compared to the bendiocarb (12% P < 0.001) and to the mixture formulations (18–22%, P < 0.05). The original Sylando 240SC formulation of chlorfenapyr was more efficacious than all other novel chlorfenapyr formulations tested. Bendiocarb induced > 80% mortality in the first month, but this declined sharply to < 20% by the third month while the mortality rates achieved with the chlorfenapyr formulations (38–46%) were persistent lasting 7–10 months. The mixtures induced significantly lower percentage mortality than chlorfenapyr-solo formulations. Wall cone bioassays only showed mortality rates that were consistent with chlorfenapyr IRS treated huts when the exposure time was increased to 2 h.ConclusionIndoor residual spraying with chlorfenapyr (Sylando® 240SC) provides moderate but prolonged control of pyrethroid-resistant malaria vectors compared to pyrethroid and bendiocarb IRS. Wall cone bioassays on chlorfenapyr-treated walls required longer exposure times of 2 h than the customary 30 min indicating that WHO guidelines on residual cone bioassays need to be more insecticide-specific.

Anopheles Species Composition and Breeding Habitat Characterisation in Chiredzi District, Zimbabwe

Malaria is a public health concern in Zimbabwe and its transmission varies geographically and seasonally, corresponding with the country's rainfall pattern. Information on density and distribution of malaria vectors, species composition, breeding sites and quality assurance of the spray programme is limited for Chiredzi District. This study investigated mosquito species composition and distribution during winter, blood meal sources of adult collections, characteristics of aquatic larval habitats and quality of spraying as well as the insecticide residual activity on sprayed walls. Anopheline larvae were collected from breeding habitats that were subsequently characterised. Adult anophelines were sampled from both inside human dwellings and outdoors. Mosquitoes collected were identified morphologically and further processed to sibling species by polymerase chain reaction (PCR) where necessary. The enzyme-linked immunosorbent assay was used to detect Plasmodium falciparum circumsporozoite proteins and multiplex PCR was used to detect blood meal source. WHO cone bioassays were conducted to monitor spray quality and insecticide residual activity on spayed walls. A total of 21 aquatic habitats were identified from which anopheline larvae were sampled. Of these, 9 (42.9 %) were classified as permanent, 7 (33.3 %) as seasonal and 5 (23.8 %) as temporal. Anopheles mosquitoes from both adult and larval collections totalled to 153, with the former collection method accounting for 69.9 % (107) and the latter for 31.1 % (46). The collected mosquitoes were identified as An. quadriannulatus and An. funestus respectively. Out of the blood-fed An. funestus females, 70 % (n = 10) had fed on human blood. None of the adult collected mosquitoes tested positive for P. falciparum circumsporozoite proteins. The aquatic habitats surveyed indicated that breeding sites were mainly man-made. Anopheles funestus was the only malaria vector recorded during this study. This species is a particularly efficient vector and its detection is a cause for concern. Furthermore, 70 % of the adult-collected An. funestus female specimens had fed on humans confirming this species' strong tendency toward anthropophagy. A more comprehensive and year-round investigation is essential to gather appropriate and relevant entomological indices on malaria transmission in Chiredzi so as to enable effective vector control.

Monitoring the durability of the long-lasting insecticidal nets Olyset\xae and PermaNet\xae 2.0 in similar use environments in Zanzibar

BackgroundMalaria transmission in Zanzibar has dramatically reduced in recent years but vector control interventions such as long-lasting insecticidal nets (LLIN) must continue to reach malaria elimination. To achieve this, the Zanzibar Malaria Elimination Programme needs actionable evidence of the durability of the LLIN brands distributed. This study compared physical and insecticidal durability of two LLIN brands: Olyset® and PermaNet© 2.0 in two similar districts on the islands of Unguja and Pemba.MethodsThis was a prospective cohort study of representative samples of households from two districts, recruited at baseline 4 months after the mass campaign. All campaign nets in these households were labelled and followed up over a period of 33 months. Primary outcome was the “proportion of nets surviving in serviceable condition” based on attrition and integrity measures and the median survival in years. The outcome for insecticidal durability was determined by bio-assay from sub-samples of campaign nets.ResultsA total of 834 campaign nets (121% of target) from 299 households were included in the study. Definite outcomes could be determined for 86% of the cohort nets in Unguja (PermaNet® 2.0) and 89% in Pemba (Olyset®). After 33 months, physical survival in serviceable condition was 55% in Unguja and 51% in Pemba. Estimated median survival was lower in Pemba at all time points with 2.3–2.7 years compared to 3.1–3.3 yeas in Unguja. Multivariable Cox proportionate hazard models confirmed the difference between brands (p < 0.0001) and identified household net-care attitude (p = 0.007) and folding of hanging nets during the day (p < 0.0001) as significant determinants, in addition to exclusive use of nets by adults (p = 0.03) and use only over a finished bedframe (p = 0.01). Optimal insecticidal effectiveness was 80% or higher for both brands at all time points when both cone bio-assays and tunnel tests were applied.ConclusionsAfter 3 years of follow-up, Olyset® LLIN showed significantly lower physical survival compared to PermaNet® 2.0 LLIN even after adjusting for other variables of net-use environment and net handling. This suggests that the differences were driven by the textile characteristics of the LLIN brands.

Indoor and outdoor residual spraying of a novel formulation of deltamethrin K-Othrine® (Polyzone) for the control of simian malaria in Sabah, Malaysia.

Since 2000, human malaria cases in Malaysia were rapidly reduced with the use of insecticides in Indoor Residual Spray (IRS) and Long-Lasting Insecticide Net (LLIN). Unfortunately, monkey malaria in humans has shown an increase especially in Sabah and Sarawak. The insecticide currently used in IRS is deltamethrin K-Othrine® WG 250 wettable granule, targeting mosquitoes that rest and feed indoor. In Sabah, the primary vector for knowlesi malaria is An. balabacensis a species known to bite outdoor. This study evaluates an alternative method, the Outdoor Residual Spray (ORS) using a novel formulation of deltamethrin K-Othrine® (PolyZone) to examine it suitability to control knowlesi malaria vector in Sabah, compared to the current method. The study was performed at seven villages in Sabah having similar type of houses (wood, bamboo and concrete). Houses were sprayed with deltamethrin K-Othrine® (PolyZone) at two different dosages, 25 mg/m2 and 30 mg/m2 and deltamethrin K-Othrine® WG 250 wettable granule at 25 mg/m2, sprayed indoor and outdoor. Residual activity on different walls was assessed using standard cone bioassay techniques. For larval surveillances, potential breeding sites were surveyed. Larvae were collected and identified, pre and post spraying. Adult survey was done using Human Landing Catch (HLC) performed outdoor and indoor. Detection of malaria parasite in adults was conducted via microscopy and molecular methods. Deltamethrin K-Othrine® (PolyZone) showed higher efficacy when sprayed outdoor. The efficacy was found varied when sprayed on different types of wall surfaces. Deltamethrin K-Othrine® (PolyZone) at 25 mg/m2 was the most effective with regards to ability to high mortality and effective knock down (KD). The vector population was reduced significantly post-spraying and reduction in breeding sites as well. The number of simian malaria infected vector, human and simian malaria transmission were also greatly reduced.

Micro- and nano-encapsulation of limonene and permethrin for mosquito repellent finishing of cotton textiles

The aim of this study was to prepare mosquito repellent textiles. To compare bio-based and synthetic mosquito repellent agents, limonene and permethrin were encapsulated with ethyl cellulose shell. Coacervation was employed, which is a simple and reproducible method for the encapsulation with high production efficiency. Morphological assessment showed that the capsules had smooth surfaces and their shape was spherical. The homogenous size distribution of the capsules was supported by laser diffraction analysis. The capsules showed narrow size distribution and the mean particle size of the optimum formulations for limonene and permethrin was 1 and 1.3 μm, respectively. The size of the produced capsules was considered suitable for textile applications. The FTIR spectra revealed the similarity of the chemical structure of capsules with shell material. Cotton fabrics were treated separately with limonene and permethrin capsules by padding method. The presence of capsules on the fabrics was determined after 20 washing cycles. Insecticide activity was evaluated against common house mosquitoes (Culex pipiens), with respect to cone bioassay of World Health Organization (WHO). Mosquitoes were tended to stay away from treated fabrics, and mortality rates of mosquitos were noted as 41% and 54% for limonene and permethrin, respectively. Although efficacy of fabrics decreased with increasing washing, the fabrics still showed repellency after 20 washing cycles. This study showed that the developed product might be used as an alternative to the other products in the market for avoiding mosquito-borne diseases.

Bio-Efficacy of Commercially Available Residual Insecticides for the Control of Aedes aegypti in Mexico.

Commercial aerosolized insecticides can be implemented as a community-based approach to targeted indoor residual spraying against Aedes aegypti, but their efficacy on pyrethroid-resistant mosquitoes has not yet been evaluated. Two commercial aerosolized products (H24 Poder Fulminante Ultra Eficaz®, carbamate, and Baygon Ultra Verde®, pyrethroid) were sprayed on common indoor surfaces e.g., cement, plywood, and cloth, and tested for their residual efficacy on susceptible and field-derived pyrethroid-resistant Ae. aegypti strains using the WHO cone bioassays. Overall, ≥80% 24-h mortality was observed for both products for at least 4 wk regardless of the mosquito strain or surface type used. H24 Poder Fulminante Ultra Eficaz showed the highest residual potency, sustaining >80% mortality for 7-wk posttreatment regardless of mosquito strain and surface type. For Baygon Ultra Verde, the mean mortality of female Ae. aegypti remained >80% for a shorter period (4-6 wk). Nonpyrethroid commercial aerosolized formulations can provide a lasting residual effect indoors compatible with the need for rapid and lasting mosquito control during outbreaks and may be suitable for community-based targeted indoor residual spraying.

Evaluation of DawaPlus 3.0 and DawaPlus 4.0, deltamethrin\u2013PBO combination nets against pyrethroid-resistant Anopheles culicifacies in experimental huts in India

BackgroundThe development of resistance in vectors is one of the major impediments for malaria control. Adding synergists to insecticides has proven to be an alternative choice for controlling resistant mosquitoes. DawaPlus 3.0 and DawaPlus 4.0 are new long-lasting insecticidal nets (LLINs) in which deltamethrin and a synergist, piperonyl butoxide (PBO) are added into filaments and their efficacy was tested against resistant malaria vector, Anopheles culicifacies in experimental huts in India.MethodsThe performance of two trial nets in terms of deterrence induced exiting, blood-feeding inhibition and mortality of An. culicifacies was compared with DawaPlus 2.0 and untreated net.ResultsThere was a significant reduction in entry, blood feeding and mortality (p < 0.05) and increase in exit rates of An. culicifacies in the treatment arms compared to untreated arm. But, both candidate LNs washed 20 times could not perform better than the washed reference net (DawaPlus 2.0). Cone bioassay results showed that all the treatment arms (both washed and unwashed) produced < 80% mortality of An. culicifacies before and after hut evaluation.ConclusionsDawaPlus 3.0 and DawaPlus 4.0 with their current specification may not be as effective as required to control the resistant vector, An. culicifacies, in east-central India.

Intensely clustered outbreak of visceral leishmaniasis (kala-azar) in a setting of seasonal migration in a village of Bihar, India

BackgroundA visceral leishmaniasis outbreak was reported from a village in a low-endemic district of Bihar, India.MethodsOutbreak investigation with house-to-house search and rapid test of kala-azar suspects and contacts was carried out. Sandfly collection and cone bio-assay was done as part of entomological study.ResultsA spatially and temporally clustered kala-azar outbreak was found at Kosra village in Sheikhpura district with 70 cases reported till December 2018. Delay of more than a year was found between diagnosis and treatment of the index case. The southern hamlet with socio-economically disadvantaged migrant population was several times more affected than rest of the village (attack rate of 19.0% vs 0.5% respectively, ORMH = 39.2, 95% CI 18.2–84.4). The median durations between onset of fever to first contact with any health services, onset to kala-azar diagnosis, diagnosis to treatment were 10 days (IQR 4–18), 30 days (IQR 17–73) and 1 day (IQR 0.5 to 3), respectively, for 50 kala-azar cases assessed till June 2017. Three-fourths of these kala-azar cases had out-of-pocket medical expenditure for their condition. Known risk factors for kala-azar such as illiteracy, poverty, belonging to socially disadvantaged community, migration, residing in kutcha houses, sleeping in rooms with unplastered walls and non-use of mosquito nets were present in majority of these cases. Only half the dwellings of the kala-azar cases were fully sprayed. Fully gravid female P. argentipes collected post indoor residual spraying (IRS) and low sandfly mortality on cone-bioassay indicated poor effectiveness of vector control.ConclusionsThere is need to focus on low-endemic areas of kala-azar. The elimination programme should implement a routine framework for kala-azar outbreak response. Complete case-finding, use of quality-compliant insecticide and coverage of all sprayable surfaces in IRS could help interrupt transmission during outbreaks.

High insecticide resistance in the major malaria vector Anopheles coluzzii in Chad Republic

BackgroundThe Sahel region of Chad Republic is a prime candidate for malaria pre-elimination. To facilitate pre-elimination efforts in this region, two populations of Anopheles coluzzii from Central Chad Republic were characterized, their insecticide resistance profile and the possible molecular mechanisms driving the resistance in the field investigated.MethodsBloodfed female Anopheles gambiae s.l. resting indoor, were collected at N’djamena and Massakory, Chad in 2018 and characterized for species composition, and infection rate was determined using the TaqMan assay. Susceptibility to various insecticides was assessed using WHO tube bioassays. Cone bioassays were conducted using various long-lasting insecticidal nets (LLINs). Results were analysed using Chi Square test. Knockdown resistance (kdr) and ace-1 markers were investigated by TaqMan genotyping.ResultsAnopheles coluzzii was the major vector found in N’djamena (100%) and Massakory (~ 94%). No Plasmodium was found in 147 bloodfed F0An. coluzzii (82 from N’djamena and 65 from Massakory). High intensity pyrethroid resistance was observed with mortalities of < 2% for permethrin, deltamethrin and etofenprox, and with < 50% and < 60% dead following exposure to 10× diagnostic doses of deltamethrin and permethrin, respectively. For both sites, < 10% mortalities were observed with DDT. Synergist bioassays with piperonylbutoxide significantly recovered pyrethroid susceptibility in Massakory populations, implicating CYP450s (mortality = 13.6% for permethrin, χ2 = 22.8, df = 1, P = 0.0006; mortality = 13.0% for deltamethrin, χ2 = 8.8, df = 1, P < 0.00031). Cone-bioassays established complete loss of efficacy of the pyrethroid-based LLINs; and a 100% recovery of susceptibility following exposure to the roof of PermaNet®3.0, containing piperonylbutoxide. Both populations were susceptible to malathion, but high bendiocarb resistance was observed in Massakory population. The absence of ace-1 mutation points to the role of metabolic resistance in the bendiocarb resistance. Both 1014F and 1014S mutations were found in both populations at around 60% and < 20% respectively. Sequencing of intron-1 of the voltage-gated sodium channel revealed a low genetic diversity suggesting reduced polymorphism.ConclusionsMultiple resistance in An. coluzzii populations from Chad highlight challenges associated with deployment of LLINs and indoor residual spraying (IRS) in the Sahel of this country. The pyrethroid-synergists LLINs (e.g. PermaNet®3.0) and organophosphate-based IRS maybe the alternatives for malaria control in this region.

Bio-efficacy and physical integrity of piperonylbutoxide coated combination net (PermaNet\xae 3.0) against pyrethroid resistant population of Anopheles gambiae s.l. and Culex quinquefasciatus mosquitoes in Ethiopia

BackgroundPermaNet® 3.0 is a deltamethrin-treated combination long-lasting insecticidal net with the addition of synergist piperonylbutoxide (PBO) on its roof section. It is designed to overcome the challenge posed by pyrethroid resistant vector populations against mainstream long-lasting insecticidal nets impregnated with pyrethroids only. The objective of this study was to determine insecticide resistance status of Anopheline and Culicine mosquitoes, to evaluate the bio-efficacy of PermaNet® 3.0 nets and to assess household factors affecting the physical integrity of PermaNet® 3.0 after 3 years of use.MethodsInsecticide susceptibility test was conducted using the WHO tube test. Bio-activity of PermaNet® 3.0 samples was evaluated using the WHO cone bioassay. Cross-sectional survey was conducted on 150 randomly selected households from two districts to determine household factors affecting net utilization. One hundred fifty PermaNet® 3.0 nets were randomly collected from the community with replacement after 3 years of deployment and physical integrity of each net was assessed.ResultsBoth Anopheles gambiae sensu lato and Culex quinquefasciatus developed resistance against permethrin and deltamethrin. However, following pre-exposure to synergist PBO the susceptibility of mosquito population increased to both permethrin (from 39% without to 92% with PBO against An. gambiae and from 28% without to 94% with PBO against Culex quinquefasciatus) and deltamethrin (from 52% without to 99% with PBO against An. gambiae and from 43% without to 98% with PBO against Culex quinquefasciatus). Eighty percent (80%) mortality was recorded in wild population of An. gambiae s.l. exposed to unused PermaNet® 3.0, but its bioactivity subsequently declined as washing frequency increased from 0 to 20. The PBO coated roof section of unused PermaNet® 3.0 resulted in higher mosquito mortality (100%) compared to the side panels without PBO (85%). House structure, cooking and washing habits, and damage due to household pests were cited as determinants associated with bed net deterioration. Bed net proportionate hole index (pHI) was ranged from 0 to 6064. Of the 150 PermaNet® 3.0 nets assessed 80, 29 and 41 were considered as ‘good’, ‘acceptable’ and ‘too torn’, respectively.ConclusionsThe bio-efficacy evaluation of PermaNet® 3.0 from Jimma area, southwestern Ethiopia showed moderate efficacy against pyrethroid resistant population of An. gambiae and Culex quinquefasciatus. Thus, NMCPs in parallel to deployment of LLINs, should implement timely insecticide resistance management and integrated vector management strategies to slowdown the evolution and further spread of insecticide resistance. Household factors such as, housing conditions, open flame fire used for cooking and rodent attack were identified as factors contributing to the observed reduced bed net physical integrity in the study area. Universal coverage of bed nets should be accompanied with community awareness creation and training on net utilization and handling.

Field evaluation of zero vector durable lining to assess its efficacy against malaria vectors and malaria transmission in tribal areas of the Balaghat district of central India.

Indoor residual spray (IRS) and long-lasting insecticidal nets are the two principal intervention methods of vector control. Zero vector durable lining (ZVDL), a relatively new vector control method, was evaluated to assess its efficacy against malaria vectors in hard to reach areas in the Balaghat district, where malaria transmission is perennial. ZVDL was installed in six experimental villages during November-December 2012. In control villages, IRS was carried out with Alphacypermethrin. Cone bioassays were performed to assess the efficacy and persistence of insecticide-treated ZVDL following WHO bioassays. The mean per man hour density of Anopheles caught during 2013 was 12.1 in experimental villages and 16.2 in control villages. No sporozoite-positive Anopheles culicifacies were found in experimental villages; however, in control villages, four sporozoite-positive A. culicifacies were found (two Plasmodium vivax and two P. falciparum). The knock-down rate of A. culicifacies was 95-100% with 100% mortality during the 24 h recovery period. Malaria declined sharply in experimental villages showing a slide positivity rate of 22.3% compared with control villages (36.4%) (p<0.05). A supplementary vector control intervention such as ZVDL has the potential to become a viable alternative to IRS in malaria endemic areas.