Detection of Invasive Anopheles stephensi Mosquitoes through Molecular Surveillance, Ghana.

The invasive Anopheles stephensi mosquito has rapidly expanded in range in Africa over the past decade. Consistent with World Health Organization guidelines, routine entomologic surveillance of malaria vectors in Accra, Ghana, now includes morphologic and molecular surveillance of An. stephensi mosquitoes. We report detection of An. stephensi mosquitoes in Ghana.

Multiple Anopheles species complicate downstream analysis and decision-making in a malaria pre-elimination area in southern Mozambique.

BACKGROUND: Different anopheline species (even within a species group/complex) can differ in their feeding and resting behaviours, which impact both malaria transmission patterns as well as the efficacy of vector control interventions. While morphological identification of sampled specimens is an important first step towards understanding species diversity and abundance, misidentification can result in the implementation of less effective vector control measures, and consequently smaller reductions in the number of local malaria cases. Focusing on southern Mozambique, a malaria pre-elimination area where malaria remains persistent, the aims of this preliminary study were to use molecular identification (CO1 and ITS2 barcoding) to (1) validate the results from the morphological identification (with a particular focus on Anopheles pharoensis and Anopheles squamosus), and (2) have a closer look at the Anopheles coustani group (which includes Anopheles tenebrosus and Anopheles ziemanni). METHODS: Female anopheline mosquitoes (n = 81) were identified morphologically and subsequently sequenced at the ribosomal DNA internal transcribed spacer region 2 (ITS2) and/or cytochrome oxidase subunit 1 (CO1) loci towards species determination. RESULTS: Out of the 62 specimens that were identified morphologically to species, 4 (6.5%) were misidentified. Regarding the An. coustani group, morphological identification showed that several members are present in southern Mozambique, including An. coustani sensu lato (s.l.), An. ziemanni and An. tenebrosus. However, based on both ITS2 and CO1 sequences, the exact species remains unknown for the latter two members until voucher sequences are available for comparison. CONCLUSION: The reason(s) for morphological misidentification of anopheline mosquitoes need to be mitigated. This is usually related to both the capacity (i.e. training) of the microscopist to identify anopheline species, and the information provided in the dichotomous identification key. As the An. coustani complex contributes to (residual) malaria transmission in sub-Saharan Africa, it may play a role in the observed persistent malaria in southern Mozambique. A better baseline characterizing of the local anophelines species diversity and behaviours will allow us to improve entomological surveillance strategies, better understand the impact of vector control on each local vector species, and identify new approaches to target those vector species.

Impact of a spatial repellent intervention on Anopheles kdr insecticide resistance allele in Sumba, Indonesia.

BACKGROUND: The emergence of insecticide resistance and outdoor transmission in malaria-endemic areas underlines the urgent need to develop innovative tools, such as spatial repellents (SR), that may circumvent this residual transmission. With limited options for effective insecticides, regular resistance monitoring is warranted for selecting and using appropriate tools. This study evaluates the pyrethroid knockdown resistance (kdr) allele before and after implementing a transfluthrin-based spatial repellent (SR) intervention in placebo-treated clusters. METHODS: This study looks at the frequency distribution of the kdr allele in Sumba Island from June 2015 to August 2018. Insecticide susceptibility tests were carried out on female Anopheles sp. aged 3-5 days against permethrin 21.5 µg/ml, deltamethrin 12.5 µg/ml, and transfluthrin 10 µg/ml using CDC bottle assay. PCR sequencing of representative samples from adult mosquito collections and insecticide tests revealed the presence of kdr mutations (L1014F and L1014S) in the VGSC gene. RESULTS: A total of 12 Anopheles species, Anopheles tesselatus, Anopheles. aconitus, Anopheles barbirostris, Anopheles kochi, Anopheles annularis, Anopheles maculatus, Anopheles sundaicus, Anopheles flavirostris, Anopheles balabacensis, Anopheles indefinitus, Anopheles subpictus, and Anopheles vagus were analysed. Anopheles vagus and An. sundaicus predominated in the larval populations. Susceptibility assays for all insecticides identified fully susceptible phenotypes in all species examined. Anopheles increasing frequency of kdr mutant alleles during the 3 year SR deployment was observed in both SR-treated and placebo areas, a statistically significant increase occurred in each arm. However, it is unclear how significant SR is in causing the increase in mutant alleles. The L1014S, knockdown resistance east type (kdr-e) allele was detected for the first time among the mosquito samples in this study. The L1014F, knockdown resistance west type (kdr-w) allele and heteroduplex form (wild-type-mutant) were found in almost all Anopheles species examined, including An. vagus, An. aconitus, An. subpictus, An. tesselatus, An. annularis, An. flavirostris and An. sundaicus. CONCLUSION: The presence of fully susceptible phenotypes over time, along with an increase in the frequency distribution of the L1014F/S mutations post-intervention, suggest drivers of resistance external to the study, including pyrethroid use in agriculture and long-lasting insecticidal nets (LLINs). However, this does not negate possible SR impacts that support resistance. More studies that enable the comprehension of possible SR-based drivers of resistance in mosquitoes need to be conducted.

Insecticide susceptibility status of Anopheles gambiae mosquitoes and the effect of pre-exposure to a piperonyl butoxide (PBO) synergist on resistance to deltamethrin in northern Namibia.

BACKGROUND: Pyrethroid-based indoor residual spraying (IRS) and long-lasting insecticidal nets (LLINs) have been employed as key vector control measures against malaria in Namibia. However, pyrethroid resistance in Anopheles mosquitoes may compromise the efficacy of these interventions. To address this challenge, the World Health Organization (WHO) recommends the use of piperonyl butoxide (PBO) LLINs in areas where pyrethroid resistance is confirmed to be mediated by mixed function oxidase (MFO). METHODS: This study assessed the susceptibility of Anopheles gambiae sensu lato (s.l.) mosquitoes to WHO tube bioassays with 4% DDT and 0.05% deltamethrin insecticides. Additionally, the study explored the effect of piperonyl butoxide (PBO) synergist by sequentially exposing mosquitoes to deltamethrin (0.05%) alone, PBO (4%) + deltamethrin (0.05%), and PBO alone. The Anopheles mosquitoes were further identified morphologically and molecularly. RESULTS: The findings revealed that An. gambiae sensu stricto (s.s.) (62%) was more prevalent than Anopheles arabiensis (38%). The WHO tube bioassays confirmed resistance to deltamethrin 0.05% in the Oshikoto, Kunene, and Kavango West regions, with mortality rates of 79, 86, and 67%, respectively. In contrast, An. arabiensis displayed resistance to deltamethrin 0.05% in Oshikoto (82% mortality) and reduced susceptibility in Kavango West (96% mortality). Notably, there was reduced susceptibility to DDT 4% in both An. gambiae s.s. and An. arabiensis from the Kavango West region. Subsequently, a subsample from PBO synergist assays in 2020 demonstrated a high proportion of An. arabiensis in Oshana (84.4%) and Oshikoto (73.6%), and 0.42% of Anopheles quadriannulatus in Oshana. Non-amplifiers were also present (15.2% in Oshana; 26.4% in Oshikoto). Deltamethrin resistance with less than 95% mortality, was consistently observed in An. gambiae s.l. populations across all sites in both 2020 and 2021. Following pre-exposure to the PBO synergist, susceptibility to deltamethrin was fully restored with 100.0% mortality at all sites in 2020 and 2021. CONCLUSIONS: Pyrethroid resistance has been identified in An. gambiae s.s. and An. arabiensis in the Kavango West, Kunene, and Oshikoto regions, indicating potential challenges for pyrethroid-based IRS and LLINs. Consequently, the data highlights the promise of pyrethroid-PBO LLINs in addressing resistance issues in the region.

Evaluating human landing catches as a measure of mosquito biting and the importance of considering additional modes of action.

Entomological evaluations of vector control tools often use human landing catches (HLCs) as a standard measure of a direct human-vector contact. However, some tools have additional characteristics, such as mortality, and HLCS are not sensitive for measuring other effects beyond landing inhibition. Therefore, additional measures may need to be considered when evaluating these tools for public health use. This study has two main aims (1) the evaluate the accuracy of HLCs as a proxy for feeding and (2) to compare the predicted reduction in vectorial capacity when we do and do not consider these additional characteristics. To achieve this, we analyse previously published semi-field data from an experiment which used HLCs and another where mosquitoes were allowed to feed in the presence of different dosages of the volatile pyrethroid spatial repellent, transfluthrin. We compare results for two mathematical models: one which only considers the reduction in feeding effect and one which also considers mortality before and after feeding (using data gathered by the aspiration of mosquitoes after the semi-field feeding/landing period and 24 h survival monitoring). These Bayesian hierarchical models are parameterised using Bayesian inference. We observe that, for susceptible mosquitoes, reduction in landing is underestimated by HLCs. For knockdown resistant mosquitoes the relationship is less clear; with HLCs sometimes appearing to overestimate this characteristic. We find HLCs tend to under-predict the relative reduction in vectorial capacity in susceptible mosquitoes while over-predicting this impact in knockdown-resistant mosquitoes. Models without secondary effects have lower predicted relative reductions in vectorial capacities. Overall, this study highlights the importance of considering additional characteristics to reduction in biting of volatile pyrethroid spatial repellents. We recommend that these are considered when evaluating novel vector control tools.

Bionomic characterization of Anopheles mosquitoes in the Ethiopian highlands and lowlands.

BACKGROUND: The protective effectiveness of vector control in malaria relies on how the implemented tools overlap with mosquito species-specific compositions and bionomic traits. In Ethiopia, targeted entomological data enabling strategic decision-making are lacking around high-risk migrant worker camps in the lowlands and resident communities in the highlands-resulting in suboptimal malaria control strategies for both populations. This study investigates spatial and temporal mosquito behavior, generating baseline evidence that will improve malaria control for both migrant workers in the lowlands and their home communities in the highlands. METHODS: Hourly Centers for Disease Control and Prevention (CDC) light trap collections were performed indoors and outdoors during the peak (October to December 2022) and minor (March to May 2023) malaria transmission seasons. These seasons coincide with the post-long rain and post-short rain seasons, respectively. Eight resident households were sampled from each of four villages in the highlands and eight households/farm structures on and near farms in four villages in the lowlands. The sampling occurred between 18:00 and 06:00. Spatiotemporal vector behaviors and hourly indoor and outdoor mosquito capture rates, used as a proxy for human biting rates, were calculated for overall catches and for individual species. Adult mosquitoes were identified using morphological keys, and a subset of samples were confirmed to species by sequencing ribosomal DNA internal transcribed spacer region 2 (ITS2) and/or mitochondrial DNA cytochrome c oxidase subunit 1 (Cox1). RESULTS: In the highlands, 4697 Anopheles mosquitoes belonging to 13 morphologically identified species were collected. The predominant species of Anopheles identified in the highlands was An. gambiae sensu lato (s.l.) (n = 1970, 41.9%), followed by An. demeilloni (n = 1133, 24.1%) and An. cinereus (n = 520, 11.0%). In the lowland villages, 3220 mosquitoes belonging to 18 morphological species were collected. Anopheles gambiae s.l. (n = 1190, 36.9%), An. pretoriensis (n = 899, 27.9%), and An. demeilloni (n = 564, 17.5%) were the predominant species. A total of 20 species were identified molecularly, of which three could not be identified to species through comparison with published sequences. In highland villages, the indoor Anopheles mosquito capture rate was much greater than the outdoor rate. This trend reversed in the lowlands, where the rate of outdoor captures was greater than the indoor rate. In both highlands and lowlands, Anopheles mosquitoes showed early biting activities in the evening, which peaked between 18:00 and 21:00, for both indoor and outdoor locations. CONCLUSIONS: The high diversity of Anopheles vectors and their variable behaviors result in a dynamic and resilient transmission system impacting both exposure to infectious bites and intervention effectiveness. This creates gaps in protection allowing malaria transmission to persist. To achieve optimal control, one-size-fits-all strategies must be abandoned, and interventions should be tailored to the diverse spatiotemporal behaviors of different mosquito populations.

Field evaluation of a volatile pyrethroid spatial repellent and etofenprox treated clothing for outdoor protection against forest malaria vectors in Cambodia.

Cambodia's goal to eliminate malaria by 2025 is challenged by persistent transmission in forest and forest fringe areas, where people are exposed to Anopheles mosquito bites during the day and night. Volatile pyrethroid spatial repellents (VPSRs) and insecticide-treated clothing (ITC) could address these gaps. This study evaluated the outdoor application of one passive transfluthrin-based VPSR, four etofenprox-ITCs paired with a picaridin topical repellent, and a combination of VPSR and ITC against wild Anopheles landing in Cambodia. A 7 × 7 Latin-square study was conducted over 49 collection nights in temporary open structures in Mondulkiri Province. All interventions substantially reduced Anopheles landing, with protective efficacy ranging from 61 to 95%. Mathematical modeling showed significant reductions in vectoral capacity, especially with the combined ITC and VPSR and VPSR alone, albeit with decreased effectiveness over time. These interventions have the potential to reduce outdoor and daytime Anopheles biting, offering valuable contributions to malaria elimination efforts in Cambodia and the Greater Mekong Subregion, contingent upon achieving effective coverage and adherence.

Rapid entomological assessment in eight high malaria endemic regencies in Papua Province revealed the presence of indoor and outdoor malaria transmissions.

Malaria in eastern Indonesia remains high despite significant reduction and elimination in other parts of the country. A rapid entomological assessment was conducted in eight high malaria endemic regencies of Papua Province, Indonesia, to expedite malaria elimination efforts in this region. This study aims to characterize specific, actionable endpoints toward understanding where and when malaria transmission is happening, where interventions may function best, and identify gaps in protection that result in continued transmission. The entomological assessment included identifying potential vectors through human landing catch (HLC), indoor morning and night resting collections, identification of larval sites through surveillance of water bodies, and vector incrimination toward understanding exposure to malaria transmission. Human landing catches (HLCs) and larval collections identified 10 Anopheles species, namely Anopheles koliensis, Anopheles punctulatus, Anopheles farauti, Anopheles hinesorum, Anopheles longirostris, Anopheles peditaeniatus, Anopheles tesselatus, Anopheles vagus, Anopheles subpictus and Anopheles kochi. The most common and abundant species found overall were An. koliensis and An. punctulatus, while An. farauti was found in large numbers in the coastal areas of Mimika and Sarmi Regencies. Vector incrimination on Anopheles collected from HLCs and night indoor resting demonstrated that An. koliensis and An. punctulatus carried Plasmodium in Keerom, Jayapura, and Sarmi Regencies. Analysis of HLCs for the most common species revealed that the An. koliensis and An. punctulatus, bite indoors and outdoors at equal rates, while An. farauti predominantly bite outdoors. Larval surveillance demonstrated that most water bodies in and surrounding residential areas contained Anopheles larvae. This study demonstrated indoor and outdoor exposure to mosquito bites and gaps in protection, enabling exposure to infectious bites in all regencies. This explains why current malaria control efforts focusing on indoor protection have failed to substantially reduce malaria incidence in the region. Optimization of insecticide-treated bed nets (ITNs), as well as installment of mosquito screens in houses, may further reduce indoor transmission. For outdoor transmission, the use of community-centric approaches to reduce or eliminate larval sources within and surrounding the village through the guidance of locally stationed entomologists, along with Social and Behavior Change mediated health education towards the local adoption of mosquito protection tools during outdoor activities, may reduce malaria transmission.

To spray or target mosquitoes another way: focused entomological intelligence guides the implementation of indoor residual spraying in southern Mozambique

Background: To eliminate malaria in southern Mozambique, the National Malaria Control Programme and its partners are scaling up indoor residual spraying (IRS) activities in two provinces, Gaza and Inhambane. An entomological surveillance planning tool (ESPT) was used to answer the programmatic question of whether IRS would be effective in target geographies, given limited information on local vector bionomics. Methods: Entomological intelligence was collected in six sentinel sites at the end of the rainy season (April-May 2018) and the beginning of the dry season (June-July 2018). The primary objective was to provide an 'entomological snapshot' by collecting question-based, timely and high-quality data within one single week in each location. Host-seeking behaviour (both indoors and outdoors) was monitored by human-baited tent traps. Indoor resting behaviour was quantified by pyrethrum spray catches and window exit traps. Results: Five different species or species groups were identified: Anopheles funestus sensu lato (s.l.) (66.0%), Anopheles gambiae s.l. (14.0%), Anopheles pharoensis (1.4%), Anopheles tenebrosus (14.1%) and Anopheles ziemanni (4.5%). Anopheles funestus sensu stricto (s.s.) was the major vector among its sibling species, and 1.9% were positive for Plasmodium falciparum infections. Anopheles arabiensis was the most abundant vector species within the An. gambiae complex, but none tested positive for P. falciparum infections. Some An. tenebrosus were positive for P. falciparum (1.3%). When evaluating behaviours that impact IRS efficacy, i.e. endophily, the known primary vector An. funestus s.s., was found to rest indoors-demonstrating at least part of its population will be impacted by the intervention if insecticides are selected to which this vector is susceptible. However, other vector species, including An. gambiae s.l., An. tenebrosus, An. pharoensis and An. ziemanni, showed exophilic and exophagic behaviours in several of the districts surveilled. Conclusion: The targeted approach to entomological surveillance was successful in collecting question-based entomological intelligence to inform decision-making about the use of IRS in specific districts. Endophilic An. funestus s.s. was documented as being the most prevalent and primary...

Fortificaçäo do leite fluido na prevençäo e tratamento da anemia carencial ferropriva em crianças menores de 4 anos / Fortification of fluid milk for the prevention and treatment of iron defficiency anemia in children under 4 years of age

Observou-se a eficácia da fortificaçäo do leite fluido com 3 mg de ferro aminoácido quelato no combate à carência de ferro em crianças menores de quatro anos. Foram acompanhadas 269 crianças que receberam, durante 12 meses, um litro de leite fortificado por dia, e que foram avaliadas a cada 6 meses de acompanhamento. Antes de se iniciar a intervençäo, a anemia estava presente em 62,3 por cento das crianças. Após 6 meses, este percentual reduziu-se a 41,8 por cento e, ao final de um ano, a 26,4 por cento. As maiores reduçöes foram detectadas nas faixas etárias de 12 a 23 meses e em menores de um ano. Das crianças que apresentavam hemoglobinas iniciais inferiores a 9,5 g/dl, 59,3 por cento recuperaram-se da anemia ao final de um ano de acompanhamento. Naquelas com hemoglobinas iniciais entre 9,5 e 10,9 g/dl, o percentual de recuperaçäo da anemia foi de 66,7 por cento. Encontrou-se, ainda, melhores evoluçöes hematológicas em crianças que ingeriam quantidades superiores a 750 ml/dia de leite fortificado, pertencentes a famílias que näo dividiam o suplemento recebido com outros membros e naquelas com apenas uma criança com menos de 5 anos no núcleo familiar. Concluiu-se pela viabilidade e eficácia da fortificaçäo do leite fluido como medida de intervençäo no combate à carência de ferro em pré-escolares