Evidence of pyrethroid resistance in Anopheles amharicus and Anopheles arabiensis from Arjo-Didessa irrigation scheme, Ethiopia

Background Indoor residual spraying and insecticide-treated nets are among the key malaria control intervention tools. However, their efficacy is declining due to the development and spread of insecticide resistant vectors. In Ethiopia, several studies reported resistance of An. arabiensis to multiple insecticide classes. However, such data is scarce in irrigated areas of the country where insecticides, pesticides and herbicides are intensively used. Susceptibility of An. gambiae s.l. to existing and new insecticides and resistance mechanisms were assessed in Arjo-Didessa sugarcane plantation area, southwestern Ethiopia. Methods Adult An. gambiae s.l. reared from larval/pupal collections of Arjo-Didessa sugarcane irrigation area and its surrounding were tested for their susceptibility to selected insecticides. Randomly selected An. gambiae s.l. (dead and survived) samples were identified to species using species-specific polymerase chain reaction (PCR) and were further analyzed for the presence of knockdown resistance (kdr) alleles using allele-specific PCR. Results Among the 214 An. gambiae s.l. samples analyzed by PCR, 89% (n = 190) were An. amharicus and 9% (n = 20) were An. arabiensis. Mortality rates of the An. gambiae s.l. exposed to deltamethrin and alphacypermethrin were 85% and 86.8%, respectively. On the other hand, mortalities against pirmiphos-methyl, bendiocarb, propoxur and clothianidin were 100%, 99%, 100% and 100%, respectively. Of those sub-samples (An. amharicus and An. arabiensis) examined for presence of kdr gene, none of them were found to carry the L1014F (West African) allelic mutation. Conclusion Anopheles amharicus and An. arabiensis from Arjo-Didessa sugarcane irrigation area were resistant to pyrethroids which might be synergized by extensive use of agricultural chemicals. Occurrence of pyrethroid resistant malaria vectors could challenge the ongoing malaria control and elimination program in the area unless resistance management strategies are implemented. Given the resistance of An. amharicus to pyrethroids, its behavior and vectorial capacity should be further investigated.

Asymptomatic and Submicroscopic Plasmodium Infections in an Area Before and During Integrated Vector Control in Homa Bay, Western Kenya

Background Long lasting insecticidal bednets (LLINs) have been the primary vector control strategy until indoor residual spraying (IRS) was added in Homa Bay and Migori Counties in western Kenya. The objective of this study was to evaluate the impact of LLINs integrated with organophosphate-based (Actellic 300 CS) IRS on the prevalence of asymptomatic and submicroscopic Plasmodium species infections in Homa Bay County. Methods Four consecutive community cross-sectional surveys for Plasmodium species infection were conducted in residents of Homa Bay County, Kenya commencing immediately before and 2 years after introduction of annual IRS. Finger-prick blood samples were obtained to prepare thick and thin smears for microscopic determination and qPCR diagnosis of Plasmodium genus, P. falciparum, P. malariae and P. ovale infection. Results Plasmodium spp. infection prevalence by microscopy was 18.5% before IRS and 14.2%, 3.3% and 1.3% after two annual rounds of IRS (χ²= 186.9, df = 3, p < 0.0001). Submicroscopic (blood smear negative, qPCR positive) parasitemia was 50.4% before IRS and 43.2%, 68.0% and 80.7% after IRS (χ²= 31.98, df = 3, p < 0.0001). Geometric mean density of P. falciparum parasitemia decreased over the 2-year study period (ANOVA, F = 28.95, df = 3, 243, p < 0.0001). The proportion of blood smear positive asymptomatic infections that included P. falciparum did not significantly change over the study period. In contrast, the proportion of asymptomatic submicroscopic P. falciparum infections trended upward following introduction of IRS (pre-IRS 48.2% versus post-IRS 41.6%, 61.3% and 75.4%; (χ²= 24.00, df = 3, p = 0.0002). Conclusions These data suggest that two annual rounds of IRS integrated with LLIN significantly reduced the prevalence of Plasmodium parasitemia, whereas the proportion of submicroscopic infections that included P. falciparum parasite increases. Strategies that aim at reducing the number of asymptomatic submicroscopic infections should be considered to diminish cryptic P. falciparum transmission and enhance malaria control.

Adult Anopheles Mosquito Distribution at a Low and High Malaria Transmission Site in Tanzania

Malaria parasites are only transmitted by female mosquitoes of the genus Anopheles; hence, the disease’s distribution is linked to that of the vector mosquitoes. As such, the goal of this study was to find out the spatial and temporal distribution of Anopheles mosquito adults in the research sites. This was a repeated cross-sectional ecological study that took place in Morogoro and Dodoma, Tanzania. Vacuum aspiration was used to collect mosquitoes both outside and inside human dwellings. All mosquito-related data was collected and entered into appropriate data collection forms. Female mosquitoes were recognized morphologically using Gillies and Coetzee morphological criteria, followed by PCR. In total, about 2742 Anopheles mosquitoes with an average collection of 18.21 ± 1.12 per day were collected outside human houses of which 1717 ( 10.51 ± 1.17 ) and 1025 ( 8.42 ± 1.41 ) were collected from Morogoro and Dodoma, respectively. Of the captured mosquitoes, 89.0%, 10.0%, and 1.0% were recognized as Anopheles arabiensis, Anopheles gambiae s.s., and Anopheles quadrianulatus, respectively. The distribution varied significantly with seasons, whereby 302 ( 4.72 ± 1.04 ) and 2440 ( 12.96 ± 1.52 ) mosquitoes were captured in the cold-dry and warm-wet season, respectively ( p < 0.0001 ). Of the captured mosquitoes, 42.33%, 16.33%, 14.96%, and 4.27 were found on the ceiling, stored junks, verandas, and barks/tree, respectively. In malaria-endemic countries, vector control forms an important component of the malaria control efforts. This study found significant variation of Anopheles mosquito abundance in time and space with Anopheles arabiensis being the most predominant malaria vector. This signifies the need to introduce mosquito control methods that will target the less anthropophilic Anopheles arabiensis or the immature aquatic stages. The study further found that underbeds, store room/piled bags, and undisturbed curtains were the most preferred resting places by mosquitoes signifying to be the most effective strategic sites for spraying insecticides during the implementation of indoor residual spraying (IRS).

Design and methods for a quasi-experimental pilot study to evaluate the impact of dual active ingredient insecticide-treated nets on malaria burden in five regions in sub-Saharan Africa

Background Vector control tools have contributed significantly to a reduction in malaria burden since 2000, primarily through insecticidal-treated bed nets (ITNs) and indoor residual spraying. In the face of increasing insecticide resistance in key malaria vector species, global progress in malaria control has stalled. Innovative tools, such as dual active ingredient (dual-AI) ITNs that are effective at killing insecticide-resistant mosquitoes have recently been introduced. However, large-scale uptake has been slow for several reasons, including higher costs and limited evidence on their incremental effectiveness and cost-effectiveness. The present report describes the design of several observational studies aimed to determine the effectiveness and cost-effectiveness of dual-AI ITNs, compared to standard pyrethroid-only ITNs, at reducing malaria transmission across a variety of transmission settings. Methods Observational pilot studies are ongoing in Burkina Faso, Mozambique, Nigeria, and Rwanda, leveraging dual-AI ITN rollouts nested within the 2019 and 2020 mass distribution campaigns in each country. Enhanced surveillance occurring in select study districts include annual cross-sectional surveys during peak transmission seasons, monthly entomological surveillance, passive case detection using routine health facility surveillance systems, and studies on human behaviour and ITN use patterns. Data will compare changes in malaria transmission and disease burden in districts receiving dual-AI ITNs to similar districts receiving standard pyrethroid-only ITNs over three years. The costs of net distribution will be calculated using the provider perspective including financial and economic costs, and a cost-effectiveness analysis will assess incremental cost-effectiveness ratios for Interceptor® G2, Royal Guard®, and piperonyl butoxide ITNs in comparison to standard pyrethroid-only ITNs, based on incidence rate ratios calculated from routine data. Conclusions Evidence of the effectiveness and cost-effectiveness of the dual-AI ITNs from these pilot studies will complement evidence from two contemporary cluster randomized control trials, one in Benin and one in Tanzania, to provide key information to malaria control programmes, policymakers, and donors to help guide decision-making and planning for local malaria control and elimination strategies. Understanding the breadth of contexts where these dual-AI ITNs are most effective and collecting robust information on factors influencing comparative effectiveness could improve uptake and availability and help maximize their impact.

Ivermectin as an endectocide may boost control of malaria vectors in India and contribute to elimination

Malaria constitutes one of the largest public health burdens faced by humanity. Malaria control has to be an efficient balance between diagnosis, treatment and vector control strategies. The World Health Organization currently recommends indoor residual spraying and impregnated bed nets as two malaria vector control methods that have shown robust and persistent results against endophilic and anthropophilic mosquito species. The Indian government launched the National Framework for Malaria Elimination in 2016 with the aim to achieve the elimination of malaria in a phased and strategic manner and to sustain a nation-wide malaria-free status by 2030. India is currently in a crucial phase of malaria elimination and novel vector control strategies maybe helpful in dealing with various challenges, such as vector behavioural adaptations and increasing insecticide resistance among the Anopheles populations of India. Ivermectin can be one such new tool as it is the first endectocide to be approved in both animals and humans. Trials of ivermectin have been conducted in endemic areas of Africa with promising results. In this review, we assess available data on ivermectin as an endectocide and propose that this endectocide should be explored as a vector control tool for malaria in India. Graphical Abstract

Monitoring of Insecticide Resistance in Anopheles culicifacies in Twelve Districts of Madhya Pradesh, Central India (2017–2019)

Background. Indoor residual spraying (IRS) and long-lasting insecticidal nets (LLINs) are malaria vector control measures used in India, but the development of insecticide resistance poses major impediments for effective vector control strategies. As per the guidelines of the National Vector Borne Disease Control Programme (NVBDCP), the study was conducted in 12 districts of Madhya Pradesh to generate data on insecticide resistance in malaria vectors. Methods. The susceptibility tests were conducted on adult An. culicifacies as per the WHO standard technique with wild-caught mosquitoes. The blood-fed female mosquitoes were exposed in 3 to 4 replicates on each occasion to the impregnated papers with specified discriminating dosages of the insecticides (DDT: 4%, malathion: 5%, deltamethrin: 0.05%, and alphacypermethrin: 0.05%), for one hour, and mortality was recorded after 24-hour holding. Results. An. culicifacies was found resistant to DDT 4% in all the 12 districts and malathion in 11 districts. The resistance to alphacypermethrin was also observed in two districts, and possible resistance was found to alphacypermethrin in seven districts and to deltamethrin in eight districts, while the vector was found susceptible to both deltamethrin and alphacypermethrin in only 3 districts. Conclusion. An. culicifacies is resistant to DDT and malathion and has emerging resistance to pyrethroids, alphacypermethrin, and deltamethrin. Therefore, regular monitoring of insecticide susceptibility in malaria vectors is needed for implementing effective vector management strategies. However, studies to verify the impact of IRS with good coverage on the transmission of disease are required before deciding on the change of insecticide in conjunction with epidemiological data.

Resting Behavior of Malaria Vectors in Ghana and Its Implication on Vector Control.

Background: In Sub-Saharan Africa, there is widespread use of long-lasting insecticidal nets (LLINs) and Indoor residual spraying (IRS) to help control the density of malaria vectors and decrease the incidence of malaria in communities. An understanding of the interactions between increased insecticide use and resting behaviour patterns of malaria mosquitoes is important for an effective vector control programme. This study was carried out to investigate the resting behavior, host preference and infection with Plasmodium falciparum of malaria vectors in Ghana in the context of increasing insecticide resistance in malaria vectors in sub-saharan Africa.Methods: Indoor and outdoor resting Anopheline mosquitoes were sampled during the dry and rainy seasons in five sites that were in 3 ecological landscapes [Sahel savannah (Kpalsogou, Pagaza, Libga), Coastal savannah (Anyakpor) and Forest (Konongo) zones] using pyrethrum spray catches (PSC), mechanical aspiration (Prokopack) for indoor collections, pit shelter and Prokopack for outdoor collections. PCR based molecular diagnostics were used to determine mosquito speciation, genotype for knockdown resistance mutations (L1014S and L1014F), G119S Ace-1 mutation, specific host blood meal origins and sporozoite infection in field collected mosquitoes.Results: Anopheles gambiae s. l. was the predominant species (89.95%, n = 1,718), followed by An. rufipes (8.48%, n=162), and An. funestus s. l. (1.57%, n = 30). Sibling species of the Anopheles gambiae revealed An. coluzzii accounted for 63% (95% CI: 57.10 – 68.91), followed by An. gambiae s. s [27% (95% CI: 21.66 – 32.55)], and An. arabiensis [9% (95% CI: 6.22 – 13.57)]. The mean resting density of An. gambiae s. l. was higher outdoors (79.63%; 1,368/1,718) than indoors (20.37%; 350/1,718) (z = -4.815, p< 0.0001). The kdr west L1014F and the Ace-1 mutations were highest in indoor resting An. coluzzii and An. gambiae in the sahel-savannah sites compared to the forest and coastal savannah sites. Overall, the blood meal analyses revealed a large proportion of the malaria vectors preferred feeding on humans (70.2 %) than animals (29.8%) in all sites. The sporozoite rates was only detected in indoor resting An. coluzzii from the sahel savannah (5.0%) and forest (2.5%) zones.Conclusion: The study reports high outdoor resting densities of An. gambiae and An. coluzzii with high kdr west mutation frequencies, and persistence of malaria transmission indoors despite the use of LLINs and IRS. Continuous monitoring of changes in resting behavior of mosquitoes and implementation of complementary malaria control interventions are needed to target outdoor resting Anopheles mosquitoes in Ghana.

Mosquito Species Composition And Insecticide Resistance Status of Anopheles Arabiensis In Itang Special Woreda, Gambella, Southwestern Ethiopia

Introduction: Anopheles arabiensis, member species of the Anopheles gambiae complex, is the primary vector of malaria widely distributed in Ethiopia. Anopheles funestus, An. pharoensis and An. nili are secondary vectors occurring with limited distribution in the country. Indoor residual spraying (IRS) and long-lasting insecticidal nets (LLINs) are pillars for the interventions against malaria control and elimination efforts in Ethiopia. However, the emergence and widespread of insecticide resistance in the major malaria vector, An. arabiensis, might compromise the efforts of the country. The aim of this study was to investigate composition of mosquito species and insecticide resistance status of An. arabiensis in Itang special woreda (district), Gambella, southwestern Ethiopia.Materials and methods: Adult mosquitoes were sampled from September 2020 to Feburary 2021 using Centers for Disease Control and Prevention (CDC) light trap and Pyrethrum Spray Catch (PSC). Moreover, mosquito larvae were also collected from different breeding sites and reared to adults to assess susceptibility status of populations of An. gambiae s.l. in the study area. Susceptibility tests were conducted on two to three days old non blood fed female An. gambiae s.l using insecticide impregnated papers with deltamethrin (0.05%), alpha-cypermethrin (0.05%), propoxur (0.1%), pirimiphos-methyl (0.25%) and bendiocarb (0.1%) following World Health Organization (WHO) standard susceptibility test procedure. Molecular diagnostics were done for the identification of member species of An. gambiae s.l and detection of knockdown resistance (kdr) allele using species specific polymerase chain reaction (PCR) and allele specific PCR. Results: In total, 468 adult mosquitoes were collected from different houses. Culex mosquitoes were the most dominant (80.4%) followed by Anopheles mosquitoes. Three species of Anopheles mosquitoes (An. coustani, An. pharoensis, and An. gambiae (s.l.)) were identified, of which An. coustani was the dominant (8.1%) species. WHO bioassay tests revealed that the populations of An. gambiae s.l in the study area are resistant against alpha-cypermethrin and deltamethrin whereas, susceptible to bendiocarb, pirimiphos-methyl and propoxur. Out of the total 86 An. gambiae s.l specimens assayed, 79 (92%) successfully amplified and identified as An. arabiensis. West African Kdr (L1014F) mutation was detected with high Kdr allele frequency ranging from 67-88%.Conclusion: The detection of target site mutation, kdr L1014F allele, coupled with the phenotypic resistance against alpha-cypermethrin and deltamethrin call for continuous resistance monitoring.

The Trend of Malaria Cases, Positivity Rate, and Determinant Factors in the Amhara Regional State, Ethiopia: A Mixed Method

Objectives. The objectives of this study were to evaluate the trend of malaria cases and test positivity rate and explore determinant factors in the Amhara Regional State, Ethiopia. Methods. A mixed study design (retrospective record data review and case study) was employed among 67 malaria officers from all zones in the region by using proportional allocation and the 1995 to 2020 malaria document review. 1995 to 2020 trend analysis was conducted using RStudio-1.2.5033. Vignette Focus Group Discussions (FGDs) were used to dig the possible factors for malaria case buildup using the purposive sampling technique, and a qualitative content analysis was used. Results. The overall mean test positivity rate (TPR) was 21.9%, and about 80% of the land of the region was malarious, and 68% of the population was at risk of malaria in the study area from the data records of 1995 to 2020. The year 2012 to 2016 had the peak confirmed malaria cases, while the year 2016 to 2018 dramatically reduced followed by an increase in 2019/2020. The vignette FGDs identified that poor performance on Larval Source Management (LSM) and net utilization, no stock of some antimalarial medicine and supply, quality of malaria diagnosis services, the low commitment of leaders, and climatic anomalies facilitated surge of the disease in 2019/2020. No real accountability at all levels, low coverage of targeted vector control interventions, resource constraint, data quality and use for informed decision making, security issues and Internally Displaced Population (IDP) in various parts of the country, and the COVID-19 pandemic were the possible causes for case buildup. Conclusions. This result revealed that the malaria incidence rate showed a remarkable decline. However, the average TPR was 21.9%. Hence, it provided the ongoing feedback, mass fever test and treatment, training to health professionals, and ongoing supportive supervision (SS) and mentorship, improved net utilization and indoor residual spraying (IRS) operation and close follow-up and conducted sensitization workshop, spot messages were transferred through mass media, and temporary case treatment and prevention centers at farm sites established may surpass the threshold of malaria.