Laboratory-based efficacy evaluation of Bacillus thuringiensis var. israelensis and temephos larvicides against larvae of Anopheles stephensi in ethiopia.

BACKGROUND: Malaria, transmitted by the bite of infective female Anopheles mosquitoes, remains a global public health problem. The presence of an invasive Anopheles stephensi, capable of transmitting Plasmodium vivax and Plasmodium falciparum parasites was first reported in Ethiopia in 2016. The ecology of An. stephensi is different from that of Anopheles arabiensis, the primary Ethiopian malaria vector, and this suggests that alternative control strategies may be necessary. Larviciding may be an effective alternative strategy, but there is limited information on the susceptibility of Ethiopian An. stephensi to common larvicides. This study aimed to evaluate the efficacy of temephos and Bacillus thuringiensis var. israelensis (Bti) larvicides against larvae of invasive An. stephensi. METHODS: The diagnostic doses of two larvicides, temephos (0.25 ml/l) and Bti (0.05 mg/l) were tested in the laboratory against the immature stages (late third to early fourth stages larvae) of An. stephensi collected from the field and reared in a bio-secure insectary. Larvae were collected from two sites (Haro Adi and Awash Subuh Kilo). For each site, three hundred larvae were tested against each insecticide (as well as an untreated control), in batches of 25. The data from all replicates were pooled and descriptive statistics prepared. RESULTS: The mortality of larvae exposed to temephos was 100% for both sites. Mortality to Bti was 99.7% at Awash and 100% at Haro Adi site. CONCLUSIONS: Larvae of An. stephensi are susceptible to temephos and Bti larvicides suggesting that larviciding with these insecticides through vector control programmes may be effective against An. stephensi in these localities.

Resistance of Anopheles stephensi to selected insecticides used for indoor residual spraying and long-lasting insecticidal nets in Ethiopia.

BACKGROUND: Malaria, transmitted by the bite of infective female Anopheles mosquitoes, remains a global public health problem. The presence of invasive Anopheles stephensi, capable of transmitting Plasmodium vivax and Plasmodium falciparum, was first reported in Ethiopia in 2016. The ecology of this mosquito species differs from that of Anopheles arabiensis, the primary malaria vector in Ethiopia. This study aimed to evaluate the efficacy of selected insecticides, which are used in indoor residual spraying (IRS) and selected long-lasting insecticidal nets (LLINs) for malaria vector control against adult An. stephensi. METHODS: Anopheles stephensi mosquitoes were collected as larvae and pupae from Awash Subah Kilo Town and Haro Adi village, Ethiopia. Adult female An. stephensi, reared from larvae and pupae collected from the field, aged 3-5 days were exposed to impregnated papers of IRS insecticides (propoxur 0.1%, bendiocarb 0.1%, pirimiphos-methyl 0.25%), and insecticides used in LLINs (alpha-cypermethrin 0.05%, deltamethrin 0.05% and permethrin 0.75%), using diagnostic doses and WHO test tubes in a bio-secure insectary at Aklilu Lemma Institute of Pathobiology, Addis Ababa University. For each test and control tube, batches of 25 female An. stephensi were used to test each insecticide used in IRS. Additionally, cone bioassay tests were conducted to expose An. stephensi from the reared population to four brands of LLINs, MAGNet™ (alpha-cypermethrin), PermaNet® 2.0 (deltamethrin), DuraNet© (alpha-cypermethrin) and SafeNet® (alpha-cypermethrin). A batch of ten sugar-fed female mosquitoes aged 2-5 days was exposed to samples taken from five positions/sides of a net. The data from all replicates were pooled and descriptive statistics were used to describe features of the data. RESULTS: All An. stephensi collected from Awash Subah Kilo Town and Haro Adi village (around Metehara) were resistant to all tested insecticides used in both IRS and LLINs. Of the tested LLINs, only MAGNet™ (alpha-cypermethrin active ingredient) caused 100% knockdown and mortality to An. stephensi at 60 min and 24 h post exposure, while all other net brands caused mortality below the WHO cut-off points (< 90%). All these nets, except SafeNet®, were collected during LLIN distribution for community members through the National Malaria Programme, in December 2020. CONCLUSIONS: Anopheles stephensi is resistant to all tested insecticides used in IRS and in the tested LLIN brands did not cause mosquito mortality as expected, except MAGNet. This suggests that control of this invasive vector using existing adult malaria vector control methods will likely be inadequate and that alternative strategies may be necessary.

Malaria among children under 10 years in 4 endemic health areas in Kisantu Health Zone: epidemiology and transmission.

BACKGROUND: The Democratic Republic of the Congo (DRC) is the second most malaria-affected country in the world with 21,608,681 cases reported in 2019. The Kongo Central (KC) Province has a malaria annual incidence of 163 cases/per 1000 inhabitants which are close to the national average of 153.4/1000. However, the malaria prevalence varies both between and within health zones in this province. The main objective of this study was to describe the epidemiology and transmission of malaria among children aged 0 to 10 years in the 4 highest endemic health areas in Kisantu Health Zone (HZ) of KC in DRC. METHODS: A community-based cross-sectional study was conducted from October to November 2017 using multi-stage sampling. A total of 30 villages in 4 health areas in Kisantu HZ were randomly selected. The prevalence of malaria was measured using a thick blood smear (TBS) and known predictors and associated outcomes were assessed. Data are described and association determinants of malaria infection were analysed. RESULTS: A total of 1790 children between 0 and 10 years were included in 30 villages in 4 health areas of Kisantu HZ. The overall prevalence in the study area according to the TBS was 14.8% (95% CI: 13.8-16.6; range: 0-53). The mean sporozoite rate in the study area was 4.3% (95% CI: 2.6-6.6). The determination of kdr-west resistance alleles showed the presence of both L1014S and L1014F with 14.6% heterozygous L1014S/L1014F, 84.4% homozygous 1014F, and 1% homozygous 1014S. The risk factors associated with malaria infection were ground or wooden floors aOR: 15.8 (95% CI: 8.6-29.2), a moderate or severe underweight: 1.5 (1.1-2.3) and to be overweight: 1.9 (95% CI: 1.3-2.7). CONCLUSION: Malaria prevalence differed between villages and health areas within the same health zone. The control strategy activities must be oriented by the variety in the prevalence and transmission of malaria in different areas. The policy against malaria regarding long-lasting insecticidal nets should be based on the evidence of metabolic resistance.

Durability of long-lasting insecticidal nets (LLINs) in Ethiopia.

BACKGROUND: The functional survival time of long-lasting insecticidal nets (LLINs), which varies across different field contexts, is critical for the successful prevention of malaria transmission. However, there is limited data on LLIN durability in field settings in Ethiopia. METHODS: A three-year longitudinal study was conducted to monitor attrition, physical integrity, and bio-efficacy and residual chemical concentration of LLINs in four regions in Ethiopia. World Health Organization (WHO) guidelines were used to determine sample size, measure physical integrity, and calculate attrition rates, and functional survival time. Yearly bio-efficacy testing was done on randomly selected LLINs. An excel tool developed by vector works project was used to calculate the median functional survival time of the LLINs. Predictors of functional survival were identified by fitting binary and multivariate cox proportional hazards model. RESULTS: A total of 3,396 LLINs were included in the analysis. A total of 3,396 LLINs were included in the analysis. By the end of 36 months, the proportion of LLINs functionally surviving was 12.9% [95% confidence interval (CI) 10.5, 15.6], the rates of attrition due to physical damage and repurposing were 48.8% [95% confidence interval (CI) 45.0, 52.6] and 13.8% [95% confidence interval (CI) 11.6, 14.6], respectively. The estimated median functional survival time was 19 months (95%CI 17, 21). Factors associated with shorter functional survival time include being in a low malaria transmission setting [Adjusted Hazards Ratio (AHR) (95%CI) 1.77 (1.22, 2.55)], rural locations [AHR (95%CI) 1.83 (1.17, 2.84)], and in a room where cooking occurs [AHR (95%CI) 1.28 (1.05, 1.55)]. Bioassay tests revealed that 95.3% (95%CI 86.4, 98.5) of the LLINs met the WHO criteria of bio-efficacy after 24 months of distribution. CONCLUSION: The LLIN survival time was shorter than the expected three years due to high attrition rates and rapid loss of physical integrity. National malaria programmes may consider, procuring more durable LLINs, educating communities on how to prevent damage of LLINs, and revising the current three-year LLIN distribution schedule to ensure sufficient protection is provided by LLINs against malaria transmission. While this paper contributes to the understanding of determinants impacting functional survival, further research is needed to understand factors for the rapid attrition rates and loss of physical integrity of LLINs in field settings.

Public health impact of the spread of Anopheles stephensi in the WHO Eastern Mediterranean Region countries in Horn of Africa and Yemen: need for integrated vector surveillance and control.

BACKGROUND: Anopheles stephensi is an efficient vector of both Plasmodium falciparum and Plasmodium vivax in South Asia and the Middle East. The spread of An. stephensi to countries within the Horn of Africa threatens progress in malaria control in this region as well as the rest of sub-Saharan Africa. METHODS: The available malaria data and the timeline for the detection of An. stephensi was reviewed to analyse the role of An. stephensi in malaria transmission in Horn of Africa of the Eastern Mediterranean Region (EMR) in Djibouti, Somalia, Sudan and Yemen. RESULTS: Malaria incidence in Horn of Africa of EMR and Yemen, increased from 41.6 in 2015 to 61.5 cases per 1000 in 2020. The four countries from this region, Djibouti, Somalia, Sudan and Yemen had reported the detection of An. stephensi as of 2021. In Djibouti City, following its detection in 2012, the estimated incidence increased from 2.5 cases per 1000 in 2013 to 97.6 cases per 1000 in 2020. However, its contribution to malaria transmission in other major cities and in other countries, is unclear because of other factors, quality of the urban malaria data, human mobility, uncertainty about the actual arrival time of An. stephensi and poor entomological surveillance. CONCLUSIONS: While An. stephensi may explain a resurgence of malaria in Djibouti, further investigations are needed to understand its interpretation trends in urban malaria across the greater region. More investment for multisectoral approach and integrated surveillance and control should target all vectors particularly malaria and dengue vectors to guide interventions in urban areas.

Entomological monitoring data driving decision-making for appropriate and sustainable malaria vector control in Côte d'Ivoire.

BACKGROUND: Entomological surveillance provides critical information on vectors for appropriate malaria vector control and strategic decision-making. The widely documented insecticide resistance of malaria vectors in Côte d'Ivoire requires that any vector control intervention deployment be driven by entomological data to optimize its effectiveness and appropriate resource allocations. To achieve this goal, this study documents the results of monthly vector surveillance and insecticide susceptibility tests conducted in 2019 and a review of all previous entomological monitoring data used to guide vector control decision making. Furthermore, susceptibility to pirimiphos-methyl and clothianidin was assessed in addition to chlorfenapyr and pyrethroids (intensity and piperonyl butoxide (PBO) synergism) tests previously reported. Vector bionomic data were conducted monthly in four sites (Sakassou, Béoumi, Dabakala and Nassian) that were selected based on their reported high malaria incidence. Adult mosquitoes were collected using human landing catches (HLCs), pyrethrum spray catches (PSCs), and human-baited CDC light traps to assess vector density, behaviour, species composition and sporozoite infectivity. RESULTS: Pirimiphos-methyl and clothianidin susceptibility was observed in 8 and 10 sites, respectively, while previous data reported chlorfenapyr (200 µg/bottle) susceptibility in 13 of the sites, high pyrethroid resistance intensity and increased mortality with PBO pre-exposure at all 17 tested sites. Anopheles gambiae sensu lato was the predominant malaria vector collected in all four bionomic sites. Vector density was relatively higher in Sakassou throughout the year with mean biting rates of 278.2 bites per person per night (b/p/n) compared to Béoumi, Dabakala and Nassian (mean of 48.5, 81.4 and 26.6 b/p/n, respectively). The mean entomological inoculation rate (EIR) was 4.44 infective bites per person per night (ib/p/n) in Sakassou, 0.34 ib/p/n in Beoumi, 1.17 ib/p/n in Dabakala and 1.02 ib/p/n in Nassian. The highest EIRs were recorded in October in Béoumi (1.71 ib/p/n) and Nassian (3.22 ib/p/n), in July in Dabakala (4.46 ib/p/n) and in May in Sakassou (15.6 ib/p/n). CONCLUSION: Based on all results and data review, the National Malaria Control Programme developed and implemented a stratified insecticide-treated net (ITN) mass distribution in 2021 considering new generation ITNs. These results also supported the selection of clothianidin-based products and an optimal spraying time for the first indoor residual spraying (IRS) campaign in Sakassou and Nassian in 2020.

Urban Onsite Sanitation Upgrades and Synanthropic Flies in Maputo, Mozambique: Effects on Enteric Pathogen Infection Risks.

Synanthropic filth flies transport enteric pathogens from feces to food, which upon consumption poses an infection risk. We evaluated the effect of an onsite sanitation intervention─including fly control measures─in Maputo, Mozambique, on the risk of infection from consuming fly-contaminated food. After enumerating flies at intervention and control sites, we cultured fecal indicator bacteria, quantified gene copies for 22 enteric pathogens via reverse transcription quantitative polymerase chain reaction (RT-qPCR), and developed quantitative microbial risk assessment (QMRA) models to estimate annual risks of infection attributable to fly-contaminated foods. We found that the intervention reduced fly counts at latrine entrances by 69% (aRR = 0.31, [0.13, 0.75]) but not at food preparation areas (aRR = 0.92, [0.33, 2.6]). Half of (23/46) of individual flies were positive for culturable Escherichia coli, and we detected ≥1 pathogen gene from 45% (79/176) of flies, including enteropathogenic E. coli (37/176), adenovirus (25/176), Giardia spp. (13/176), and Trichuris trichiura (12/176). We detected ≥1 pathogen gene from half the flies caught in control (54%, 30/56) and intervention compounds (50%, 17/34) at baseline, which decreased 12 months post-intervention to 43% (23/53) at control compounds and 27% (9/33) for intervention compounds. These data indicate flies as a potentially important mechanical vector for enteric pathogen transmission in this setting. The intervention may have reduced the risk of fly-mediated enteric infection for some pathogens, but infrequent detection resulted in wide confidence intervals; we observed no apparent difference in infection risk between groups in a pooled estimate of all pathogens assessed (aRR = 0.84, [0.61, 1.2]). The infection risks posed by flies suggest that the design of sanitation systems and service delivery should include fly control measures to prevent enteric pathogen transmission.

Association of Reduced Long-Lasting Insecticidal Net Efficacy and Pyrethroid Insecticide Resistance With Overexpression of CYP6P4, CYP6P3, and CYP6Z1 in Populations of Anopheles coluzzii From Southeast Côte d'Ivoire.

BACKGROUND: Resistance to major public health insecticides in Côte d'Ivoire has intensified and now threatens the long-term effectiveness of malaria vector control interventions. METHODS: This study evaluated the bioefficacy of conventional and next-generation long-lasting insecticidal nets (LLINs), determined resistance profiles, and characterized molecular and metabolic mechanisms in wild Anopheles coluzzii from Southeast Côte d'Ivoire in 2019. RESULTS: Phenotypic resistance was intense: >25% of mosquitoes survived exposure to 10 times the doses of pyrethroids required to kill susceptible populations. Similarly, the 24-hour mortality rate with deltamethrin-only LLINs was very low and not significantly different from that with an untreated net. Sublethal pyrethroid exposure did not induce significant delayed vector mortality effects 72 hours later. In contrast, LLINs containing the synergist piperonyl butoxide, or new insecticides clothianidin and chlorfenapyr, were highly toxic to A. coluzzii. Pyrethroid-susceptible A. coluzzii were significantly more likely to be infected with malaria, compared with those that survived insecticidal exposure. Pyrethroid resistance was associated with significant overexpression of CYP6P4, CYP6P3, and CYP6Z1. CONCLUSIONS: Study findings raise concerns regarding the operational failure of standard LLINs and support the urgent deployment of vector control interventions incorporating piperonyl butoxide, chlorfenapyr, or clothianidin in areas of high resistance intensity in Côte d'Ivoire.

The potential impact of Anopheles stephensi establishment on the transmission of Plasmodium falciparum in Ethiopia and prospective control measures.

BACKGROUND: Sub-Saharan Africa has seen substantial reductions in cases and deaths due to malaria over the past two decades. While this reduction is primarily due to an increasing expansion of interventions, urbanisation has played its part as urban areas typically experience substantially less malaria transmission than rural areas. However, this may be partially lost with the invasion and establishment of Anopheles stephensi. A. stephensi, the primary urban malaria vector in Asia, was first detected in Africa in 2012 in Djibouti and was subsequently identified in Ethiopia in 2016, and later in Sudan and Somalia. In Djibouti, malaria cases have increased 30-fold from 2012 to 2019 though the impact in the wider region remains unclear. METHODS: Here, we have adapted an existing model of mechanistic malaria transmission to estimate the increase in vector density required to explain the trends in malaria cases seen in Djibouti. To account for the observed plasticity in An. stephensi behaviour, and the unknowns of how it will establish in a novel environment, we sample behavioural parameters in order to account for a wide range of uncertainty. This quantification is then applied to Ethiopia, considering temperature-dependent extrinsic incubation periods, pre-existing vector-control interventions and Plasmodium falciparum prevalence in order to assess the potential impact of An. stephensi establishment on P. falciparum transmission. Following this, we estimate the potential impact of scaling up ITN (insecticide-treated nets)/IRS (indoor residual spraying) and implementing piperonyl butoxide (PBO) ITNs and larval source management, as well as their economic costs. RESULTS: We estimate that annual P. falciparum malaria cases could increase by 50% (95% CI 14-90) if no additional interventions are implemented. The implementation of sufficient control measures to reduce malaria transmission to pre-stephensi levels will cost hundreds of millions of USD. CONCLUSIONS: Substantial heterogeneity across the country is predicted and large increases in vector control interventions could be needed to prevent a major public health emergency.

Identification of a rapidly-spreading triple mutant for high-level metabolic insecticide resistance in Anopheles gambiae provides a real-time molecular diagnostic for antimalarial intervention deployment.

Studies of insecticide resistance provide insights into the capacity of populations to show rapid evolutionary responses to contemporary selection. Malaria control remains heavily dependent on pyrethroid insecticides, primarily in long lasting insecticidal nets (LLINs). Resistance in the major malaria vectors has increased in concert with the expansion of LLIN distributions. Identifying genetic mechanisms underlying high-level resistance is crucial for the development and deployment of resistance-breaking tools. Using the Anopheles gambiae 1000 genomes (Ag1000g) data we identified a very recent selective sweep in mosquitoes from Uganda which localized to a cluster of cytochrome P450 genes. Further interrogation revealed a haplotype involving a trio of mutations, a nonsynonymous point mutation in Cyp6p4 (I236M), an upstream insertion of a partial Zanzibar-like transposable element (TE) and a duplication of the Cyp6aa1 gene. The mutations appear to have originated recently in An. gambiae from the Kenya-Uganda border, with stepwise replacement of the double-mutant (Zanzibar-like TE and Cyp6p4-236 M) with the triple-mutant haplotype (including Cyp6aa1 duplication), which has spread into the Democratic Republic of Congo and Tanzania. The triple-mutant haplotype is strongly associated with increased expression of genes able to metabolize pyrethroids and is strongly predictive of resistance to pyrethroids most notably deltamethrin. Importantly, there was increased mortality in mosquitoes carrying the triple-mutation when exposed to nets cotreated with the synergist piperonyl butoxide (PBO). Frequencies of the triple-mutant haplotype remain spatially variable within countries, suggesting an effective marker system to guide deployment decisions for limited supplies of PBO-pyrethroid cotreated LLINs across African countries.

Insecticidal effects of some selected plant extracts against Anopheles stephensi (Culicidae: Diptera).

BACKGROUND: The use of synthetic insecticides against mosquitoes may lead to resistance development and potential health hazards in humans and the environment. Consequently, a paradigm needs to shift towards the alternative use of botanical insecticides that could strengthen an insecticide resistance management programme. This study aimed to assess the insecticidal effects aqueous, hexane, and methanol crude leaf extracts of Calpurnia aurea, Momordica foetida, and Zehneria scabra on an insectary colony of Anopheles stephensi larvae and adults. METHODS: Fresh leaves of C. aurea, M. foetida and Z. scabra were collected and dried, then separately ground to powder. Powdered leaves of test plants were extracted using sonication with aqueous, hexane, and methanol solvents. The extracts were concentrated, and a stock solution was prepared. For comparison, Temephos (Abate®) and control solutions (a mixture of water and emulsifier) were used as the positive and negative controls, respectively. Different test concentrations for the larvae and the adults were prepared and tested according to WHO (2005) and CDC (2010) guidelines to determine lethal concentration (LC) values. Mortality was observed after 24 h exposure. The statistical analyses were performed using Statistical Package for the Social Sciences (SPSS) software (Kruskal-Wallis test) and R software (a generalized linear model was used to determine LC50 and LC90 values of the extracts). RESULTS: The lowest LC50 values were observed in aqueous extracts of M. foetida followed by Z. scabra extract and C. aurea leaves at 34.61, 35.85, and 38.69 ppm, respectively, against the larvae. Larval mortality was not observed from the hexane extracts and negative control, while the standard larvicide (temephos) achieved 100% mortality. Further, the adulticidal efficacy was greatest for aqueous extract of Z. scabra with LC50 = 176.20 ppm followed by aqueous extract of C. aurea (LC50 = 297.75 ppm). CONCLUSION: The results suggest that the leaf extracts of the three test plants have the potential of being used for the control of vector An. stephensi larvae and adult instead of synthetic mosquitocides. Further studies need to be conducted to identify the active ingredients and their mode of action.

Evaluation of an accelerometer-based monitor for detecting bed net use and human entry/exit using a machine learning algorithm.

BACKGROUND: Distribution of long-lasting insecticidal bed nets (LLINs) is one of the main control strategies for malaria. Improving malaria prevention programmes requires understanding usage patterns in households receiving LLINs, but there are limits to what standard cross-sectional surveys of self-reported LLIN use can provide. This study was designed to assess the performance of an accelerometer-based approach for measuring a range of LLIN use behaviours as a proof of concept for more granular LLIN-use monitoring over longer time periods. METHODS: This study was carried out under controlled conditions from May to July 2018 in Liverpool, UK. A single accelerometer was affixed to the side panel of an LLIN and participants carried out five LLIN use behaviours: (1) unfurling a net; (2) entering an unfurled net; (3) lying still as if sleeping; (4) exiting from under a net; and, (5) folding up a net. The randomForest package in R, a supervised non-linear classification algorithm, was used to train models on 20-s epochs of tagged accelerometer data. Models were compared in a validation dataset using overall accuracy, sensitivity and specificity, receiver operating curves and the area under the curve (AUC). RESULTS: The five-category model had overall accuracy of 82.9% in the validation dataset, a sensitivity of 0.681 for entering a net, 0.632 for exiting, 0.733 for net down, and 0.800 for net up. A simplified four-category model, combining entering/exiting a net into one category had accuracy of 94.8%, and increased sensitivity for net down (0.756) and net up (0.829). A further simplified three-category model, identifying sleeping, net up, and a combined net down/enter/exit category had accuracy of 96.2% (483/502), with an AUC of 0.997 for net down and 0.987 for net up. Models for detecting entering/exiting by adults were significantly more accurate than for children (87.8% vs 70.0%; p < 0.001) and had a higher AUC (p = 0.03). CONCLUSIONS: Understanding how LLINs are used is crucial for planning malaria prevention programmes. Accelerometer-based systems provide a promising new methodology for studying LLIN use. Further work exploring accelerometer placement, frequency of measurements and other machine learning approaches could make these methods even more accurate in the future.

An experimental hut study evaluating the impact of pyrethroid-only and PBO nets alone and in combination with pirimiphos-methyl-based IRS in Ethiopia.

BACKGROUND: Pyrethroid resistance observed in populations of malaria vectors is widespread in Ethiopia and could potentially compromise the effectiveness of insecticide-based malaria vector control interventions. In this study, the impact of combining indoor residual spraying (IRS) and insecticide-treated nets (ITNs) on mosquito behaviour and mortality was evaluated using experimental huts. METHODS: A Latin Square Design was employed using six experimental huts to collect entomological data. Human volunteers slept in huts with different types of nets (pyrethroid-only net, PBO net, and untreated net) either with or without IRS (Actellic 300CS). The hut with no IRS and an untreated net served as a negative control. The study was conducted for a total of 54 nights. Both alive and dead mosquitoes were collected from inside nets, in the central rooms and verandah the following morning. Data were analysed using Stata/SE 14.0 software package (College Station, TX, USA). RESULTS: The personal protection rate of huts with PermaNet® 2.0 alone and PermaNet® 3.0 alone was 33.3% and 50%, respectively. The mean killing effect of huts with PermaNet® 2.0 alone and PermaNet® 3.0 alone was 2% and 49%, respectively. Huts with PermaNet® 2.0 alone and PermaNet® 3.0 alone demonstrated significantly higher excito-repellency than the control hut. However, mosquito mortality in the hut with IRS + untreated net, hut with IRS + PermaNet® 2.0 and hut with IRS + PermaNet® 3.0 were not significantly different from each other (p > 0.05). Additionally, pre-exposure of both the susceptible Anopheles arabiensis laboratory strain and wild Anopheles gambiae sensu lato to PBO in the cone bioassay tests of Actellic 300CS sprayed surfaces did not reduce mosquito mortality when compared to mortality without pre-exposure to PBO. CONCLUSION: Mosquito mortality rates from the huts with IRS alone were similar to mosquito mortality rates from the huts with the combination of vector control intervention tools (IRS + ITNs) and mosquito mortality rates from huts with PBO nets alone were significantly higher than huts with pyrethroid-only nets. The findings of this study help inform studies to be conducted under field condition for decision-making for future selection of cost-effective vector control intervention tools.

Combined Epidemiologic and Entomologic Survey to Detect Urban Malaria Transmission, Guinea, 2018.

Malaria incidence is generally lower in cities than rural areas. However, reported urban malaria incidence may not accurately reflect the level of ongoing transmission, which has potentially large implications for prevention efforts. To guide mosquito net distribution, we assessed the extent of malaria transmission in Conakry, Guinea, in 2018. We found evidence of active malaria transmission.

Anopheles stephensi Mosquitoes as Vectors of Plasmodium vivax and falciparum, Horn of Africa, 2019.

Anopheles stephensi mosquitoes, efficient vectors in parts of Asia and Africa, were found in 75.3% of water sources surveyed and contributed to 80.9% of wild-caught Anopheles mosquitoes in Awash Sebat Kilo, Ethiopia. High susceptibility of these mosquitoes to Plasmodium falciparum and vivax infection presents a challenge for malaria control in the Horn of Africa.

Adaptation of ELISA detection of Plasmodium falciparum and Plasmodium vivax circumsporozoite proteins in mosquitoes to a multiplex bead-based immunoassay.

BACKGROUND: Plasmodium spp. sporozoite rates in mosquitoes are used to better understand malaria transmission intensity, the relative importance of vector species and the impact of interventions. These rates are typically estimated using an enzyme-linked immunosorbent assay (ELISA) utilizing antibodies against the circumsporozoite protein of Plasmodium falciparum, Plasmodium vivax VK210 (P. vivax210) or P. vivax VK247 (P. vivax247), employing assays that were developed over three decades ago. The ELISA method requires a separate assay plate for each analyte tested and can be time consuming as well as requiring sample volumes not always available. The bead-based multiplex platform allows simultaneous measurement of multiple analytes and may improve the lower limit of detection for sporozoites. METHODS: Recombinant positive controls for P. falciparum, P. vivax210 and P. vivax247 and previously developed circumsporozoite (cs) ELISA antibodies were used to optimize conditions for the circumsporozoite multiplex bead assay (csMBA) and to determine the detection range of the csMBA. After optimizing assay conditions, known amounts of sporozoites were used to determine the lower limit of detection for the csELISA and csMBA and alternate cut-off measures were applied to demonstrate how cut-off criteria can impact lower limits of detection. Sporozoite rates from 1275 mosquitoes collected in Madagascar and 255 mosquitoes collected in Guinea were estimated and compared using the established csELISA and newly optimized csMBA. All mosquitoes were tested (initial test), and those that were positive were retested (retest). When sufficient sample volume remained, an aliquot of homogenate was boiled and retested (boiled retest), to denature any heat-unstable cross-reactive proteins. RESULTS: Following optimization of the csMBA, the lower limit of detection was 25 sporozoites per mosquito equivalent for P. falciparum, P. vivax210 and P. vivax247 whereas the lower limits of detection for csELISA were found to be 1400 sporozoites for P. falciparum, 425 for P. vivax210 and 1650 for P. vivax247. Combined sporozoite rates after re-testing of samples that initially tested positive for Madagascar mosquitoes by csELISA and csMBA were 1.4 and 10.3%, respectively, and for Guinea mosquitoes 2% by both assays. Boiling of samples followed by csMBA resulted in a decrease in the Madagascar sporozoite rate to 2.8-4.4% while the Guinea csMBA sporozoite rate remained at 2.0%. Using an alternative csMBA cut-off value of median fluorescence intensity (MFI) of 100 yielded a sporozoite rate after confirmational testing of 3.7% for Madagascar samples and 2.0% for Guinea samples. Whether using csMBA or csELISA, the following steps may help minimize false positives: specimens are appropriately stored and bisected anterior to the thorax-abdomen junction, aliquots of homogenate are boiled and retested following initial testing, and an appropriate cut-off value is determined. CONCLUSIONS: The csMBA is a cost-comparable and time saving alternative to the csELISA and may help eliminate false negatives due to a lower limit of detection, thus increasing sensitivity over the csELISA. The csMBA expands the potential analyses that can be done with a small volume of sample by allowing multiplex testing where analytes in addition to P. falciparum, P. vivax210 and P. vivax247 can be added following optimization.

Malaria in migrant agricultural workers in western Ethiopia: entomological assessment of malaria transmission risk.

BACKGROUND: Ethiopia has made great strides in malaria control over the last two decades. However, this progress has not been uniform and one concern has been reported high rates of malaria transmission in large agricultural development areas in western Ethiopia. Improved vector control is one way this transmission might be addressed, but little is known about malaria vectors in this part of the country. METHODS: To better understand the vector species involved in malaria transmission and their behaviour, human landing collections were conducted in Dangur woreda, Benishangul-Gumuz, between July and December 2017. This period encompasses the months with the highest rain and the peak mosquito population. Mosquitoes were identified to species and tested for the presence of Plasmodium sporozoites. RESULTS: The predominant species of the Anopheles collected was Anopheles arabiensis (1,733; i.e. 61.3 % of the entire Anopheles), which was also the only species identified with sporozoites (Plasmodium falciparum and Plasmodium vivax). Anopheles arabiensis was collected as early in the evening as 18:00 h-19:00 h, and host-seeking continued until 5:00 h-6:00 h. Nearly equal numbers were collected indoors and outdoors. The calculated entomological inoculation rate for An. arabiensis for the study period was 1.41 infectious bites per month. More An. arabiensis were collected inside and outside worker's shelters than in fields where workers were working at night. CONCLUSIONS: Anopheles arabiensis is likely to be the primary vector of malaria in the agricultural development areas studied. High rates of human biting took place inside and outdoor near workers' residential housing. Improved and targeted vector control in this area might considerably reduce malaria transmission.

The effect of long-lasting insecticidal nets (LLINs) physical integrity on utilization.

BACKGROUND: In Ethiopia, despite improvements in coverage and access, utilization of long-lasting insecticidal nets (LLINs) remains a challenge. Different household-level factors have been identified as associated with LLIN use. However, the contribution of LLIN physical integrity to their utilization is not well investigated and documented. This study aimed to assess the association between the physical integrity of LLINs and their use. METHODS: This study employed a nested case-control design using secondary data from the Ethiopian LLIN durability monitoring study conducted from May 2015 to June 2018. LLINs not used the night before the survey were identified as cases, while those used the previous night were categorized as controls. The physical integrity of LLINs was classified as no holes, good, acceptable, and torn using the proportionate hole index (pHI). A Generalized Estimating Equation (GEE) model was used to assess and quantify the association between LLIN physical integrity and use. The model specifications included binomial probabilistic distribution, logit link, exchangeable correlation matrix structure, and robust standard errors. The factors included in the model were selected first by fitting binary regression, and then by including all factors that showed statistical significance at P-value less than 0.25 and conceptually relevant variables into the multivariate regression model. RESULTS: A total of 5277 observations fulfilled the inclusion criteria. Out of these 1767 observations were cases while the remaining 3510 were controls. LLINs that were in torn physical condition had higher odds (AOR [95% CI] = 1.76 [1.41, 2.19]) of not being used compared to LLINs with no holes. Other factors that showed significant association included the age of the LLIN, sleeping place type, washing status of LLINs, perceptions towards net care and repair, LLIN to people ratio, economic status, and study site. CONCLUSION AND RECOMMENDATION: LLINs that have some level of physical damage have a relatively higher likelihood of not being used. Community members need to be educated about proper care and prevention of LLIN damage to delay the development of holes as long as possible and use available LLINs regularly.

Malaria prevention and treatment in migrant agricultural workers in Dangur district, Benishangul-Gumuz, Ethiopia: social and behavioural aspects.

BACKGROUND: Sixty percent of the Ethiopia population is at risk of malaria, with the highest prevalence reported in Gambella (6%) and Benishangul-Gumuz (3%) regions. Within these regions are large agricultural developments with high numbers of seasonal migrant workers. The migrant workers are believed to be at increased risk for malaria infection due to their poor living conditions and outdoor activities, but there is little information on their specific behaviours and health risks. This study was conducted to address this gap. METHODS: Quantitative observations were conducted from September to December 2017 in the Benishangul-Gumuz Region. The nightly routines of mobile migrant workers were observed every month for 4 consecutive months. The study team collected quantitative data including nocturnal behavioural observations of worker living conditions, malaria prevention efforts, and work activities and surveys of worker representatives. Qualitative data was collected from migrant workers, farm managers and local health providers using focus group discussions and semi-structured interviews. RESULTS: Migrant workers arrived in the study area during the peak malaria transmission season and the workers in focus groups reported repeated cases of malaria during their stay on the farms. Overall, less than a quarter of the migrant workers were sleeping under a mosquito net by midnight in all 4 observation months. Some work activities also took place outdoors at night. The study additionally found a lack of access to malaria prevention and treatment at the farms and challenges in utilizing local public health facilities. CONCLUSIONS: There is a need to better address malaria prevention and treatment needs among migrant workers in Ethiopia through outreach from existing healthcare infrastructure and within the farms themselves. This will help prevent malaria transmission both within this population and prevent transmission of malaria back to home communities in lower burden areas in Ethiopia.

An update on the distribution, bionomics, and insecticide susceptibility of Anopheles stephensi in Ethiopia, 2018-2020.

BACKGROUND: Anopheles stephensi, an invasive malaria vector, was first detected in Africa nearly 10 years ago. After the initial finding in Djibouti, it has subsequently been found in Ethiopia, Sudan and Somalia. To better inform policies and vector control decisions, it is important to understand the distribution, bionomics, insecticide susceptibility, and transmission potential of An. stephensi. These aspects were studied as part of routine entomological monitoring in Ethiopia between 2018 and 2020. METHODS: Adult mosquitoes were collected using human landing collections, pyrethrum spray catches, CDC light traps, animal-baited tent traps, resting boxes, and manual aspiration from animal shelters. Larvae were collected using hand-held dippers. The source of blood in blood-fed mosquitoes and the presence of sporozoites was assessed through enzyme-linked immunosorbent assays (ELISA). Insecticide susceptibility was assessed for pyrethroids, organophosphates and carbamates. RESULTS: Adult An. stephensi were collected with aspiration, black resting boxes, and animal-baited traps collecting the highest numbers of mosquitoes. Although sampling efforts were geographically widespread, An. stephensi larvae were collected in urban and rural sites in eastern Ethiopia, but An. stephensi larvae were not found in western Ethiopian sites. Blood-meal analysis revealed a high proportion of blood meals that were taken from goats, and only a small proportion from humans. Plasmodium vivax was detected in wild-collected An. stephensi. High levels of insecticide resistance were detected to pyrethroids, carbamates and organophosphates. Pre-exposure to piperonyl butoxide increased susceptibility to pyrethroids. Larvae were found to be susceptible to temephos. CONCLUSIONS: Understanding the bionomics, insecticide susceptibility and distribution of An. stephensi will improve the quality of a national response in Ethiopia and provide additional information on populations of this invasive species in Africa. Further work is needed to understand the role that An. stephensi will have in Plasmodium transmission and malaria case incidence. While additional data are being collected, national programmes can use the available data to formulate and operationalize national strategies against the threat of An. stephensi.