Fine-scale mapping of urban malaria exposure under data scarcity: an approach centred on vector ecology.

BACKGROUND: Although malaria transmission has experienced an overall decline in sub-Saharan Africa, urban malaria is now considered an emerging health issue due to rapid and uncontrolled urbanization and the adaptation of vectors to urban environments. Fine-scale hazard and exposure maps are required to support evidence-based policies and targeted interventions, but data-driven predictive spatial modelling is hindered by gaps in epidemiological and entomological data. A knowledge-based geospatial framework is proposed for mapping the heterogeneity of urban malaria hazard and exposure under data scarcity. It builds on proven geospatial methods, implements open-source algorithms, and relies heavily on vector ecology knowledge and the involvement of local experts. METHODS: A workflow for producing fine-scale maps was systematized, and most processing steps were automated. The method was evaluated through its application to the metropolitan area of Dakar, Senegal, where urban transmission has long been confirmed. Urban malaria exposure was defined as the contact risk between adult Anopheles vectors (the hazard) and urban population and accounted for socioeconomic vulnerability by including the dimension of urban deprivation that is reflected in the morphology of the built-up fabric. Larval habitat suitability was mapped through a deductive geospatial approach involving the participation of experts with a strong background in vector ecology and validated with existing geolocated entomological data. Adult vector habitat suitability was derived through a similar process, based on dispersal from suitable breeding site locations. The resulting hazard map was combined with a population density map to generate a gridded urban malaria exposure map at a spatial resolution of 100 m. RESULTS: The identification of key criteria influencing vector habitat suitability, their translation into geospatial layers, and the assessment of their relative importance are major outcomes of the study that can serve as a basis for replication in other sub-Saharan African cities. Quantitative validation of the larval habitat suitability map demonstrates the reliable performance of the deductive approach, and the added value of including local vector ecology experts in the process. The patterns displayed in the hazard and exposure maps reflect the high degree of heterogeneity that exists throughout the city of Dakar and its suburbs, due not only to the influence of environmental factors, but also to urban deprivation. CONCLUSIONS: This study is an effort to bring geospatial research output closer to effective support tools for local stakeholders and decision makers. Its major contributions are the identification of a broad set of criteria related to vector ecology and the systematization of the workflow for producing fine-scale maps. In a context of epidemiological and entomological data scarcity, vector ecology knowledge is key for mapping urban malaria exposure. An application of the framework to Dakar showed its potential in this regard. Fine-grained heterogeneity was revealed by the output maps, and besides the influence of environmental factors, the strong links between urban malaria and deprivation were also highlighted.

Updates on malaria epidemiology and profile in Cabo Verde from 2010 to 2019: the goal of elimination.

BACKGROUND: Located in West Africa, Cabo Verde is an archipelago consisting of nine inhabited islands. Malaria has been endemic since the settlement of the islands during the sixteenth century and is poised to achieve malaria elimination in January 2021. The aim of this research is to characterize the trends in malaria cases from 2010 to 2019 in Cabo Verde as the country transitions from endemic transmission to elimination and prevention of reintroduction phases. METHODS: All confirmed malaria cases reported to the Ministry of Health between 2010 and 2019 were extracted from the passive malaria surveillance system. Individual-level data available included age, gender, municipality of residence, and the self-reported countries visited if travelled within the past 30 days, therby classified as imported. Trends in reported cases were visualized and multivariable logistic regression used to assess risk factors associated with a malaria case being imported and differences over time. RESULTS: A total of 814 incident malaria cases were reported in the country between 2010 and 2019, the majority of which were Plasmodium falciparum. Overall, prior to 2017, when the epidemic occurred, 58.1% (95% CI 53.6-64.6) of infections were classified as imported, whereas during the post-epidemic period, 93.3% (95% CI 86.9-99.7) were imported. The last locally acquired case was reported in January 2018. Imported malaria cases were more likely to be 25-40 years old (AOR: 15.1, 95% CI 5.9-39.2) compared to those under 15 years of age and more likely during the post-epidemic period (AOR: 56.1; 95% CI 13.9-225.5) and most likely to be reported on Sao Vicente Island (AOR = 4256.9, 95% CI = 260-6.9e+4) compared to Boavista. CONCLUSIONS: Cabo Verde has made substantial gains in reducing malaria burden in the country over the past decade and are poised to achieve elimination in 2021. However, the high mobility between the islands and continental Africa, where malaria is still highly endemic, means there is a constant risk of malaria reintroduction. Characterization of imported cases provides useful insight for programme and enables better evidence-based decision-making to ensure malaria elimination can be sustained.

Evaluation of the residual efficacy of indoor residual spraying with bendiocarb (FICAM WP 80) in six health districts in Senegal.

BACKGROUND: From 2011 to 2014, an indoor residual spray (IRS) programme for malaria vectors control was implemented in six health districts in Senegal. The main objective of the present study was to evaluate the efficacy of bendiocarb (FICAM WP 80) sprayed on different wall surfaces and its impact on malaria vectors. The entomological monitoring activities were carried out monthly in five treated sentinel villages and one control untreated village in each district. METHODS: The residual efficacy of bendiocarb applied at a dosage of 0.4 g/sq m was monitored for a period up to 9 months post-IRS using WHO cone bioassay method. This assay consisted to expose 2-5 days old unfed susceptible Anopheles coluzzii females to sprayed walls for a period of 30 min. The mortality rates after 24 h post-exposure were estimated and compared between the different types of walls sprayed in each sentinel village. RESULTS: The results showed that the residual efficacy varied between the different sprayed walls, from one sentinel village to another and between the different campaigns. The FICAM had a residual efficacy of 3-6 months post-IRS on mud and cement wall surfaces. In some cases, the observed mortality rates were much higher than those reported elsewhere particularly during the first campaign in all the six districts. CONCLUSIONS: The FICAM was found to be effective with a residual efficacy varying from 3 to 6 months. If the quality of the IRS application is excluded as a possible explanation of the short efficacy duration, the results suggest at least two rounds of treatments in order to cover the rainy season that lasts 5 to 6 months in the area. Such treatments could be carried out before the intensification of the rains in July and August in order to better cover the transmission period that occurs between late August and October in the area.

Knowledge, attitudes and practices about malaria in Cabo Verde: a country in the pre-elimination context.

BACKGROUND: Malaria in Cape Verde is unstable, with a sporadic and seasonal transmission of low endemicity. In this sense, the community perceptions regarding malaria transmission, their attitudes and practices against the disease are very important to understand and to better develop the best strategical policies to achieve malaria elimination goal. This study aim to assess the knowledge, attitudes and practices (KAP) of Cape Verdean population about malaria, a country in the elimination step of disease. METHODS: A cross-sectional malaria KAP Survey was performed at the household level. A structured open questionnaire was developed and applied to residents of randomly selected households from 5 islands and 15 municipalities in Cape Verde. Correlation analyses were performed using a logistic regression model to determine the factors that are associated with the complete knowledge of the population about malaria. RESULTS: A total of 1953 fully completed questionnaires were analysed, with majority of questionnaires administered in Santiago island (68.3%), mainly in the capital city of Praia, 38.43%. About 88% of the population knew the correct form of transmission, 96% had knowledge that the entire population is at risk of malaria and identified the main symptoms. Regarding the attitudes, 58% seek treatment atthe nearest health structure upon the apparition of the symptoms, 64% in the first 24 h and 88% within the first 48 h. More than 97% have heard about mosquito nets but only 19% used it. In practice, 53% use coils, 45% rely on household sprays and 43% have benefited from IRS. About 90% received information about malaria from media, mainly the TV and the radio (83 and 43%, respectively). In summary, 54% of the population has complete knowledge of the disease. CONCLUSION: The population of Cape Verde has a high level of knowledge about malaria, including its transmission, main symptoms and preventive and control measures. However, some gaps and misunderstandings have been noticed and contribute to the insufficient community involvement in actions against malaria. Therefore, is necessary to increase the knowledge of the population, leading to their full ownership and participation in community actions to contribute to the malaria elimination in the country.

First report of natural Wolbachia infection in wild Anopheles funestus population in Senegal.

BACKGROUND: Until very recently, Anopheles were considered naturally unable to host Wolbachia, an intracellular bacterium regarded as a potential biological control tool. Their detection in field populations of Anopheles gambiae sensu lato, suggests that they may also be present in many more anopheline species than previously thought. RESULTS: Here, is reported the first discovery of natural Wolbachia infections in Anopheles funestus populations from Senegal, the second main malaria vector in Africa. Molecular phylogeny analysis based on the 16S rRNA gene revealed at least two Wolbachia genotypes which were named wAnfu-A and wAnfu-B, according to their close relatedness to the A and B supergroups. Furthermore, both wAnfu genotypes displayed high proximity with wAnga sequences previously described from the An. gambiae complex, with only few nucleotide differences. However, the low prevalence of infection, together with the difficulties encountered for detection, whatever method used, highlights the need to develop an effective and sensitive Wolbachia screening method dedicated to anopheline. CONCLUSIONS: The discovery of natural Wolbachia infection in An. funestus, another major malaria vector, may overcome the main limitation of using a Wolbachia-based approach to control malaria through population suppression and/or replacement.

Achievement of malaria pre-elimination in Cape Verde according to the data collected from 2010 to 2016.

BACKGROUND: Malaria, despite being preventable and treatable, continues to be a major public health problem worldwide. The archipelago nation of Cape Verde is in a malaria pre-elimination phase with the highest potential to achieve the target goal of elimination in 2020. METHODS: Nationwide malaria epidemiological data were obtained from the Cape Verde health information system that includes the individual malaria case notification system from all of the country's health structures. Each case is reported to the surveillance service then to the National Malaria Control Programme, which allowed for compilation in the national malaria case database. The database was analysed to assess the origin of the malaria cases, and incidence was calculated from 2010 to 2016 by sex and age. The health centre, health district and month of diagnosis were evaluated, as well as the sex and the age of the patients, allowing a direct descriptive analysis of national data to provide an up-to-date malaria epidemiological profile of the country. Malaria cases were classified as imported or indigenous, and then, geographical analyses were performed using a unique Geographical National Code with Quantum Geographic Information System 2.16.2 software to map the cases by municipalities. The overall temporal evolution of cases was analysed to assess their monthly and yearly variations from 2010 to 2016. RESULTS: Malaria is unstable in Cape Verde, with inter-annual variation and the majority of infections occurring in adult males (> 20 years). The indigenous cases are restricted to Santiago (96%) and Boavista (4%), while imported cases were recorded in all the nine inhabited islands, originating from neighbouring countries with ongoing malaria transmission; from Lusophone countries (25% from Angola, 25% from Guinea-Bissau), followed by the Republic of Senegal (12%) and Equatorial Guinea (10%). In 2010-2012, more imported (93 cases) than indigenous cases (26 cases) were observed; conversely, in 2013 and 2014, more indigenous cases (49) than imported cases (42) were reported. In 2015 there were 20 imported cases and only 7 indigenous cases. Finally, in 2016, there were 47 indigenous cases and 28 imported cases. The mapping of cases by municipality and country of origin was possible with GIS analyses. CONCLUSION: While Cape Verde remains on track to achieve malaria elimination by 2020 owing to the reduction of the annual incidence to below 0.1%, the country still records cases of indigenous and imported malaria. However, the indigenous cases are exclusively confined to the Santiago and Boavista islands, while the imported cases recorded nationwide originate only from the African continent, mainly from adult men from the Lusophone countries. Cape Verde needs to target interventions to remove residual foci on Santiago and Boavista islands to reduce malaria lethality to zero and prevent its reintroduction from African countries via transmission across the archipelago. Cape Verde is a good example of local authority's commitment to tackle malaria and work towards its elimination by strengthening the health and surveillance systems.

Entomological impact of indoor residual spraying with pirimiphos-methyl: a pilot study in an area of low malaria transmission in Senegal.

BACKGROUND: Scaling-up of effective anti-malarial control strategies in Central-West region of Senegal has resulted in the sharp decline in malaria prevalence in this area. However, despite these strategies, residual malaria transmission has been observed in some villages (hot spots). The objective of this study was to assess the impact of indoor residual spraying (IRS) with pirimiphos-methyl on malaria transmission in hot spot areas. METHODS: The malaria vector population dynamics were monitored in each of the six selected villages (4 of which used IRS, 2 were unsprayed control areas) using overnight human landing catches (HLC) and pyrethrum spray catches (PSC). The host source of blood meals from freshly fed females collected using PSC was identified using the direct ELISA method. Females caught through HLC were tested by ELISA for the detection of Plasmodium falciparum circumsporozoite protein and Anopheles gambiae complex was identified using PCR. RESULTS: Preliminary data shown that the densities of Anopheles populations were significantly lower in the sprayed areas (179/702) compared to the control. Overall, malaria transmission risk was 14 times lower in the intervention zone (0.94) compared to the control zone (12.7). In the control areas, three Anopheles species belonging to the Gambiae complex (Anopheles arabiensis, Anopheles coluzzii and Anopheles melas) maintained the transmission, while only An. coluzzii was infective in the sprayed areas. CONCLUSION: The preliminary data from this pilot study showed that IRS with the CS formulation of pirimiphos-methyl is likely very effective in reducing malaria transmission risk. However, additional studies including further longitudinal entomological surveys as well as ecological and ethological and genetical characterization of vectors species and their populations are needed to better characterize the entomological impact of indoor residual spraying with pirimiphos-methyl in the residual transmission areas of Senegal.

Predicting the age of field Anopheles mosquitoes using mass spectrometry and deep learning.

Mosquito-borne diseases like malaria are rising globally, and improved mosquito vector surveillance is needed. Survival of Anopheles mosquitoes is key for epidemiological monitoring of malaria transmission and evaluation of vector control strategies targeting mosquito longevity, as the risk of pathogen transmission increases with mosquito age. However, the available tools to estimate field mosquito age are often approximate and time-consuming. Here, we show a rapid method that combines matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry with deep learning for mosquito age prediction. Using 2763 mass spectra from the head, legs, and thorax of 251 field-collected Anopheles arabiensis mosquitoes, we developed deep learning models that achieved a best mean absolute error of 1.74 days. We also demonstrate consistent performance at two ecological sites in Senegal, supported by age-related protein changes. Our approach is promising for malaria control and the field of vector biology, benefiting other disease vectors like Aedes mosquitoes.

Distribution and dynamics of Anopheles gambiae s.l. larval habitats in three Senegalese cities with high urban malaria incidence.

Urban malaria has become a challenge for most African countries due to urbanization, with increasing population sizes, overcrowding, and movement into cities from rural localities. The rapid expansion of cities with inappropriate water drainage systems, abundance of water storage habitats, coupled with recurrent flooding represents a concern for water-associated vector borne diseases, including malaria. This situation could threaten progress made towards malaria elimination in sub-Saharan countries, including Senegal, where urban malaria has presented as a threat to national elimination gains. To assess drivers of urban malaria in Senegal, a 5-month study was carried out from August to December 2019 in three major urban areas and hotspots for malaria incidence (Diourbel, Touba, and Kaolack) including the rainy season (August-October) and partly dry season (November-December). The aim was to characterize malaria vector larval habitats, vector dynamics across both seasons, and to identify the primary eco- environmental entomological factors contributing to observed urban malaria transmission. A total of 145 Anopheles larval habitats were found, mapped, and monitored monthly. This included 32 in Diourbel, 83 in Touba, and 30 in Kaolack. The number of larval habitats fluctuated seasonally, with a decrease during the dry season. In Diourbel, 22 of the 32 monitored larval habitats (68.75%) were dried out by December and considered temporary, while the remaining 10 (31.25%) were classified as permanent. In the city of Touba 28 (33.73%) were temporary habitats, and of those 57%, 71% and 100% dried up respectively by October, November, and December. However, 55 (66.27%) habitats were permanent water storage basins which persisted throughout the study. In Kaolack, 12 (40%) permanent and 18 (60%) temporary Anopheles larval habitats were found and monitored during the study. Three malaria vectors (An. arabiensis, An. pharoensis and An. funestus s.l.) were found across the surveyed larval habitats, and An. arabiensis was found in all three cities and was the only species found in the city of Diourbel, while An. arabiensis, An. pharoensis, and An. funestus s.l. were detected in the cities of Touba and Kaolack. The spatiotemporal observations of immature malaria vectors in Senegal provide evidence of permanent productive malaria vector larval habitats year-round in three major urban centers in Senegal, which may be driving high urban malaria incidence. This study aimed to assess the presence and type of anopheline larvae habitats in urban areas. The preliminary data will better inform subsequent detailed additional studies and seasonally appropriate, cost-effective, and sustainable larval source management (LSM) strategies by the National Malaria Control Programme (NMCP).

Characterization of the swarming behavior of Anopheles coluzzii and Anopheles gambiae (Diptera: Culicidae) populations in a hybrid zone of Senegal.

Anopheles gambiae and Anopheles coluzzii, often found in sympatry and synchronous, have undergone a premating reproductive isolation across their distribution range. However, in the Western coast of Africa, unexpected hybridization zones have been observed, and little is known about swarming behavior of these cryptic taxa. Here, we characterized the swarming behavior of An. coluzzii and An. gambiae to investigate its role in the high hybridization level in Senegal. The study was conducted in the south and central Senegal during the 2018 rainy season. Mating swarms of malaria vectors were surveyed at sunset and collected using an insect net. Meanwhile, indoor resting populations of malaria vectors were collected by pyrethrum spray catches. Upon collection, specimens were identified morphologically, and then members of the An. gambiae complex were identified at the species level by polymerase chain reaction (PCR). An. gambiae swarmed mainly over bare ground, whereas An. coluzzii were found swarming above various objects creating a dark-light contrast with the bare ground. The swarms height varied from 0.5 to 2.5 m. Swarming starting time was correlated with sunset whatever the months for both species, and generally lasted about 10 min. No mixed swarm of An. gambiae and An. coluzzii was found even in the high hybridization area. These results indicated a premating isolation between An. coluzzii and An. gambiae. However, the high hybridization rate in the sympatric area suggests that heterogamous mating is occurring, thus stressing the need for further extensive studies.

Urban malaria vector bionomics and human sleeping behavior in three cities in Senegal.

BACKGROUND: Malaria is endemic in Senegal, with seasonal transmission, and the entire population is at risk. In recent years, high malaria incidence has been reported in urban and peri-urban areas of Senegal. An urban landscape analysis was conducted in three cities to identify the malaria transmission indicators and human behavior that may be driving the increasing malaria incidence occurring in urban environments. Specifically, mosquito vector bionomics and human sleeping behaviors including outdoor sleeping habits were assessed to guide the optimal deployment of targeted vector control interventions. METHODS: Longitudinal entomological monitoring using human landing catches and pyrethrum spray catches was conducted from May to December 2019 in Diourbel, Kaolack, and Touba, the most populous cities in Senegal after the capital Dakar. Additionally, a household survey was conducted in randomly selected houses and residential Koranic schools in the same cities to assess house structures, sleeping spaces, sleeping behavior, and population knowledge about malaria and vector control measures. RESULTS: Of the 8240 Anopheles mosquitoes collected from all the surveyed sites, 99.4% (8,191) were An. gambiae s.l., and predominantly An. arabiensis (99%). A higher number of An. gambiae s.l. were collected in Kaolack (77.7%, n = 6496) than in Diourbel and Touba. The overall mean human biting rate was 14.2 bites per person per night (b/p/n) and was higher outdoors (15.9 b/p/n) than indoors (12.5 b/p/n). The overall mean entomological inoculation rates ranged from 3.7 infectious bites per person per year (ib/p/y) in Diourbel to 40.2 ib/p/y in Kaolack. Low anthropophilic rates were recorded at all sites (average 35.7%). Of the 1202 households surveyed, about 24.3% of household members slept outdoors, except during the short rainy season between July and October, despite understanding how malaria is transmitted and the vector control measures used to prevent it. CONCLUSION: Anopheles arabiensis was the primary malaria vector in the three surveyed cities. The species showed an outdoor biting tendency, which represents a risk for the large proportion of the population sleeping outdoors. As all current vector control measures implemented in the country target endophilic vectors, these data highlight potential gaps in population protection and call for complementary tools and approaches targeting outdoor biting malaria vectors.

Evolution of the Ace-1 and Gste2 Mutations and Their Potential Impact on the Use of Carbamate and Organophosphates in IRS for Controlling Anopheles gambiae s.l., the Major Malaria Mosquito in Senegal.

Widespread of insecticide resistance amongst the species of the Anopheles gambiae complex continues to threaten vector control in Senegal. In this study, we investigated the presence and evolution of the Ace-1 and Gste2 resistance genes in natural populations of Anopheles gambiae s.l., the main malaria vector in Senegal. Using historical samples collected from ten sentinel health districts, this study focused on three different years (2013, 2017, and 2018) marking the periods of shift between the main public health insecticides families (pyrethroids, carbamates, organophosphates) used in IRS to track back the evolutionary history of the resistance mutations on the Ace-1 and Gste2 loci. The results revealed the presence of four members of the Anopheles gambiae complex, with the predominance of An. arabiensis followed by An. gambiae, An. coluzzii, and An. gambiae-coluzzii hybrids. The Ace-1 mutation was only detected in An. gambiae and An. gambiae-coluzzii hybrids at low frequencies varying between 0.006 and 0.02, while the Gste2 mutation was found in all the species with a frequency ranging between 0.02 and 0.25. The Ace-1 and Gste2 genes were highly diversified with twenty-two and thirty-one different haplotypes, respectively. The neutrality tests on each gene indicated a negative Tajima's D, suggesting the abundance of rare alleles. The presence and spread of the Ace-1 and Gste2 resistance mutations represent a serious threat to of the effectiveness and the sustainability of IRS-based interventions using carbamates or organophosphates to manage the widespread pyrethroids resistance in Senegal. These data are of the highest importance to support the NMCP for evidence-based vector control interventions selection and targeting.

Evolution of the Pyrethroids Target-Site Resistance Mechanisms in Senegal: Early Stage of the Vgsc-1014F and Vgsc-1014S Allelic Frequencies Shift.

The evolution and spread of insecticide resistance mechanisms amongst malaria vectors across the sub-Saharan Africa threaten the effectiveness and sustainability of current insecticide-based vector control interventions. However, a successful insecticide resistance management plan relies strongly on evidence of historical and contemporary mechanisms circulating. This study aims to retrospectively determine the evolution and spread of pyrethroid resistance mechanisms among natural Anopheles gambiae s.l. populations in Senegal. Samples were randomly drawn from an existing mosquito sample, collected in 2013, 2017, and 2018 from 10 sentinel sites monitored by the Senegalese National Malaria Control Programme (NMCP). Molecular species of An. gambiae s.l. and the resistance mutations at the Voltage-gated Sodium Channel 1014 (Vgsc-1014) locus were characterised using PCR-based assays. The genetic diversity of the Vgsc gene was further analyzed by sequencing. The overall species composition revealed the predominance of Anopheles arabiensis (73.08%) followed by An. gambiae s.s. (14.48%), Anopheles coluzzii (10.94%) and Anopheles gambiae-coluzii hybrids (1.48%). Both Vgsc-1014F and Vgsc-1014S mutations were found in all studied populations with a spatial variation of allele frequencies from 3% to 90%; and 7% to 41%, respectively. The two mutations have been detected since 2013 across all the selected health districts, with Vgsc-L1014S frequency increasing over the years while Vgsc-1014F decreasing. At species level, the Vgsc-1014F and Vgsc-1014S alleles were more frequent amongst An. gambiae s.s. (70%) and An. arabiensis (20%). The Vgsc gene was found to be highly diversified with eight different haplotypes shared between Vgsc-1014F and Vgsc-1014S. The observed co-occurrence of Vgsc-1014F and Vgsc-1014S mutations suggest that pyrethroid resistance is becoming a widespread phenomenon amongst malaria vector populations, and the NMCP needs to address this issue to sustain the gain made in controlling malaria.

Development of a multiplex qPCR-based approach for the diagnosis of Dirofilaria immitis, D. repens and Acanthocheilonema reconditum.

BACKGROUND: Dirofilaria immitis, D. repens and Acanthocheilonema reconditum are the main causative agents of zoonotic canine filariosis. METHODS: We developed a combined multiplex approach for filaria and Wolbachia detection using the 28S-based pan-filarial and 16S-based pan-Wolbachia qPCRs, respectively, involving a fast typing method of positive samples using triplex qPCR targeting A. reconditum, D. immitis and D. repens, and a duplex qPCR targeting Wolbachia of D. immitis and D. repens. The approach was complemented by a duplex qPCR for the differential diagnosis of heartworms (D. immitis and Angiostrongylus vasorum) and pan-filarial cox1 and pan-Wolbachia ftsZ PCRs to identify other filarial parasites and their Wolbachia, respectively. A total of 168 canine blood and sera samples were used to validate the approach. Spearman's correlation was used to assess the association between filarial species and the strain of Wolbachia. Positive samples for both the heartworm antigen-test after heating sera and at least one DNA-positive for D. immitis and its Wolbachia were considered true positive for heartworm infection. Indeed, the presence of D. repens DNA or that of its Wolbachia as well as A. reconditum DNA indicates true positive infections. RESULTS: The detection limit for Wolbachia and filariae qPCRs ranged from 5 × 10-1 to 1.5 × 10-4 mf/ml of blood. When tested on clinical samples, 29.2% (49/168) tested positive for filariae or Wolbachia DNA. Filarial species and Wolbachia genotypes were identified by the combined multiplex approach from all positive samples. Each species of Dirofilaria was significantly associated with a specific genotype of Wolbachia. Compared to the true positives, the approach showed excellent agreement (k = 0.98-1). Unlike D. immitis DNA, no A. vasorum DNA was detected by the duplex qPCR. The immunochromatographic test for heartworm antigen showed a substantial (k = 0.6) and a weak (k = 0.15) agreements before and after thermal pre-treatment of sera, respectively. CONCLUSIONS: The proposed approach is a reliable tool for the exploration and diagnosis of occult and non-occult canine filariosis. The current diagnosis of heartworm disease based on antigen detection should always be confirmed by qPCR essays. Sera heat pre-treatment is not effective and strongly discouraged.

Role of plants in the transmission of Asaia sp., which potentially inhibit the Plasmodium sporogenic cycle in Anopheles mosquitoes.

Biological control against malaria and its transmission is currently a considerable challenge. Plant-associated bacteria of the genus Asaia are frequently found in nectarivorous arthropods, they thought to have a natural indirect action on the development of plasmodium in mosquitoes. However, virtually nothing is known about its natural cycle. Here, we show the role of nectar-producing plants in the hosting and dissemination of Asaia. We isolated Asaia strains from wild mosquitoes and flowers in Senegal and demonstrated the transmission of the bacteria from infected mosquitoes to sterile flowers and then to 26.6% of noninfected mosquitoes through nectar feeding. Thus, nectar-producing plants may naturally acquire Asaia and then colonize Anopheles mosquitoes through food-borne contamination. Finally, Asaia may play an indirect role in the reduction in the vectorial capacity of Anopheles mosquitoes in a natural environment (due to Plasmodium-antagonistic capacities of Asaia) and be used in the development of tools for Asaia-based paratransgenetic malaria control.

Hymenopteran Parasitoids of Hard Ticks in Western Africa and the Russian Far East.

Some parasitoids of the genus Ixodiphagus (Hymenoptera, Chalcidoidea: Encyrtidae) are well-known natural enemies of ticks. In this study, we investigate the occurrence of parasitoid wasps in adult hard ticks from Western Africa (Côte d'Ivoire and Senegal) and Far Eastern Europe (Russia) using molecular methods. The morphological identification allowed the classification of 785 collected specimens of six species of ticks: Rhipicephalus (Boophilus) microplus (41%), Ixodes persulcatus (33%), Dermacentor silvarum (11%), Haemaphysalis concinna (7%), Amblyomma variegatum (5%), and Haemaphysalis japonica (3%). The newly developed MALDI-TOF MS protocol identified tick species in spite of their different storage (dried or in 70% ethanol) conditions for a long period. Molecular screening of ticks by a new standard PCR system developed in this study revealed the presence of parasitoid wasp DNA in 3% (28/785) of analyzed ticks. Ixodiphagus hookeri was detected in 86% (24/28) of infested ticks, including 13 I. persulcatus, 9 R (B) microplus, and one H. concinna and D. silvarum. While an unidentified parasitoid wasp species from the subfamily Aphidiinae and Braconidae family was detected in the remaining 14% (4/28) infested ticks. These infested ticks were identified as I. persulcatus. Our findings highlight the need for further studies to clarify the species diversity of parasitoid infesting ticks by combining molecular and morphological features. The novel molecular and MALDI-TOF MS protocols could be effective tools for the surveillance and characterization of these potential bio-control agents of ticks.

Mapping the breeding sites of Anopheles gambiae s. l. in areas of residual malaria transmission in central western Senegal.

Despite the deployment of several effective control interventions in central-western Senegal, residual malaria transmission is still occurring in some hotspots. To better tailor targeted control actions, it is critical to unravel the underlying environmental and geographical factors that cause the persistence infection in hotspot villages. "Hotspots villages" were defined in our study as those reporting more than six indigenous malaria cases during the previous year. A total of ten villages, including seven hotspots and three non-hotspots, were surveyed. All potential mosquito breeding sites identified in and around the ten study villages were regularly monitored between 2013 and 2017. Monitoring comprised the detection of anopheline larvae and the collection of epidemiological, hydrogeological, topographical, and biogeographical data. The number of larval breeding sites described and monitored during the study period ranged from 50 to 62. Breeding sites were more numerous in hotspot sites in each year of monitoring, with 90.3% (56/62) in 2013, 90.9% (50/55) in 2014, 90.3% (56/62) in 2015 and 86% (43/50) in 2017 (Fisher exact test; p = 1). In the non-hotspot areas, the data for the same years were, respectively, 9.7% (6/62), 9.1% (5/55), 9.7% (6/62) and 14% (7/50) (p = 1). The Hotspot villages were characterized mostly by saline or moderately saline hydro-morphic and halomorphic soils allowing water retention and a potential larval breeding sites. By contrast, non-hotspot villages were characterized mainly by a high proportion of extremely permeable sandy-textured soils, which due to their porosity had low water retention. The annual number of confirmed malaria cases was correlated with the frequency and extent of breeding sites. Malaria cases were significantly more frequent in the hamlets located near breeding sites of An. gambiae s.l., gradually decreasing with increasing remoteness. This study shows that the characteristics of larval breeding sites, as measured by their longevity, stability, proximity to human habitation, and their positivity in Anopheles larvae are likely determining factors in the persistence of malaria hotspots in central-western Senegal. The results of this study shed more light on the environmental factors underlying the residual transmission and should make it possible to better target vector control interventions for malaria elimination in west-central Senegal.

Multiple insecticide resistance target sites in adult field strains of An. gambiae (s.l.) from southeastern Senegal.

BACKGROUND: High coverage of long-lasting insecticidal nets (LLINs) and indoor residual spraying (IRS) are the cornerstones of vector control strategy in Senegal where insecticide resistance by the target vectors species is a great of concern. This study explores insecticide susceptibility profile and target-site mutations mechanisms within the Anopheles gambiae complex in southeastern Senegal. METHODS: Larvae of Anopheles spp. were collected in two sites from southeastern Senegal Kedougou and Wassadou/Badi in October and November 2014, and reared until adult emergence. Wild F0 adult mosquitoes were morphologically identified to species. Susceptibility of 3-5-day-old An. gambiae (s.l.) samples to 11 insecticides belonging to the four insecticide classes was assessed using the WHO insecticide susceptibility bioassays. Tested samples were identified using molecular techniques and insecticide resistance target-site mutations (kdr, ace-1 and rdl) were determined. RESULTS: A total of 3742 An. gambiae (s.l.) were exposed to insecticides (2439 from Kedougou and 1303 from Wassadou-Badi). Tests with pyrethroid insecticides and DDT showed high level of resistance in both Kedougou and Wassadou/Badi. Resistance to pirimiphos-methyl and malathion was not detected while resistance to bendoicarb and fenitrothion was confirmed in Kedougou. Of the 745 specimens of An. gambiae (s.l.) genotyped, An. gambiae (s.s.) (71.6%) was the predominant species, followed by An. arabiensis (21.7%), An. coluzzii (6.3%) and hybrids (An. gambiae (s.s.)/An. coluzzii; 0.4%). All target site mutations investigated (Vgsc-1014F, Vgsc-1014S, Ace-1 and Rdl) were found at different frequencies in the species of the Anopheles gambiae complex. Vgsc-1014F mutation was more frequent in An. gambiae (s.s.) and An. coluzzii than An. arabiensis. Vgsc-1014S was present in An. gambiae (s.l.) populations in Wassadou but not in Kedougou. Ace-1 and rdl mutations were more frequent in An. gambiae (s.s.) in comparison to An. arabiensis and An. coluzzii. CONCLUSIONS: Resistance to all the four insecticide classes tested was detected in southeastern Senegal as well as all target site mutations investigated were found. Data will be used by the national Malaria Control Programme.

The prevalence of glucose-6-phosphate dehydrogenase deficiency in the Cape Verdean population in the context of malaria elimination.

Cabo Verde aims to eliminate malaria by 2020. In the country, Plasmodium falciparum had been the main parasite responsible for indigenous cases and primaquine is the first line treatment of cases and for radical cure. However, the lack of knowledge of the national prevalence of glucose-6-phosphate dehydrogenase (G6PD) deficiency may be one of the constraints to the malaria elimination process. Hence, this first study determines the prevalence of G6PD deficiency (G6PDd) in the archipelago. Blood samples were collected from patients who voluntarily agreed to participate in the study, in the health facilities of eight municipalities on four islands, tested with G6PD CareStart ™ deficiency Rapid Diagnosis Test (RDT). All subjects found to be G6PDd by RDT then underwent enzyme quantification by spectrophotometry. Descriptive statistics and inferences were done using SPSS 22.0 software. A total of 5.062 blood samples were collected, in majority from female patients (78.0%) and in Praia (35.6%). The RDT revealed the prevalence of G6PD deficiency in 2.5% (125/5062) of the general population, being higher in males (5.6%) than in females (1,6%). The highest G6PDd prevalence was recorded in São Filipe, Fogo, (5.4%), while in Boavista no case was detected. The G6PDd activity quantification shown a higher number of partially deficient and deficient males (respectively n = 26 and n = 22) compared to females (respectively n = 18 and n = 7), but more normal females (n = 35) than males (n = 11). According to the WHO classification, most of the G6PDd cases belongs to the class V (34.5%), while the Classes II and I were the less represented with respectively 5.8% and zero cases. This study in Cabo Verde determined the G6PDd prevalence in the population, relatively low compared to other African countries. Further studies are needed to characterize and genotyping the G6PD variants in the country.

Vector bionomics and malaria transmission in an area of sympatry of An. arabiensis, An. coluzzii and An. gambiae.

Despite extensive genetic studies on their variability and differentiation, few is known about the specific and relative role of An. coluzzii, An. gambiae and An. arabiensis in areas of sympatry. Indeed, their behavioral dissimilarities and divergent population dynamics can impact on malaria transmission level and intensity. This study was undertaken in four sympatric sites belonging to two different ecosystems with differential insecticide pressure to study the bionomics of these species and their relative role in malaria transmission. Mosquitoes were collected monthly from July to December 2011 when landing on human volunteers and by pyrethrum spray catches. Specimens belonging to the An. gambiae complex were further identified using molecular tools. Plasmodium falciparum infection and blood-feeding preferences were studied using the ELISA techniques. Overall, the three species were in sympatry in each of the four sites with the predominance of An. gambiae. Mosquito populations' dynamics varied temporally depending on the rainy season for each zone. The anthropophilic rates varied between 45.7 and 78.1% for An. arabiensis, 81.8 and 100% for An. coluzzii and 80 and 96.7% for An. gambiae. Plasmodium infection rates were higher in An. gambiae (range: 2.17%-6.54%) while for An. arabiensis and An. coluzzii it varied respectively between 0-1.24% and 0-3.66%. Malaria transmission occured in each of the four sites both indoors and outdoors and was due mainly to An. gambiae. An. arabiensis and An. coluzzii played a limited role due both to a low anthropophilic rate and a lower biting rate for An. coluzzii in comparison with An. gambiae. This study showed that, while present in sympatric areas, species from the An. gambiae complex could exhibit differential involvement in malaria transmission. Even less involved in malaria transmission, the occurrence of ecological and environmental changes tending to a good adaptation of An. coluzzii could lead to a great risk for malaria transmission in time and space in human populations.