An Entomological Investigation during a Recent Rift Valley Fever Epizootic/Epidemic Reveals New Aspects of the Vectorial Transmission of the Virus in Madagascar.

A Rift Valley fever (RVF) outbreak occurred in at least five regions of Madagascar in 2021. The aim of this study was to provide an overview of the richness, abundance, ecology, and trophic preferences of mosquitoes in the Mananjary district and to investigate the distribution of mosquitoes that were RT-PCR-positive for RVFV. Three localities were prospected from 26 April to 4 May 2021, using light traps, BG-Sentinel traps baited with an artificial human odor, Muirhead-Thomson pit traps, and indoor pyrethroid spray catches. A total of 2806 mosquitoes belonging to at least 26 species were collected. Of 512 monospecific pools of mosquitoes tested with real-time RT-PCR, RVFV was detected in 37 pools representing 10 mosquito species. The RVFV-positive species were as follows: Aedes albopictus, Ae. argenteopunctatus, Anopheles coustani, An. gambiae s.l., An. mascarensis, An. squamosus/cydippis, Culex antennatus, Cx. decens, Cx. Tritaeniorhynchus, and Uranotaenia spp. Of the 450 tested engorged females, 78.7% had taken a blood meal on humans, 92.9% on cattle, and 71.6% had taken mixed (human-cattle) blood meals. This investigation suggests the potential role of mosquitoes in RVFV transmission within this epizootic/epidemic context and that the human populations at the three study sites were highly exposed to mosquitoes. Therefore, the use of impregnated mosquito nets as an appropriate prevention method is recommended.

The Re-Emergence of Rift Valley Fever in Mananjary District, Madagascar in 2021: A Call for Action.

An epizootic of rift valley fever (RVF) was suspected on 21 February 2021 in various districts of Madagascar, with a lab confirmation on 1 April 2021. A cross-sectional survey aiming to detect cases of RVF in humans and to study the circulation of rift valley fever virus (RVFV) in animals was conducted from 22 April to 4 May 2021 in the district of Mananjary. Blood samples from cattle and humans were tested using serological and molecular techniques. In cattle, the circulation of RVFV was confirmed between 5 February and 4 May 2021. The positivity rates of anti-RVFV IgG and IgM were 60% and 40%, respectively. In humans, the circulation of RVFV was observed from 1 April to 5 May 2021. The positivity rate of RVFV was estimated to be 11.7% by combining the results of the molecular and serological approaches. Of the 103 individuals who agreed to participate in the survey, 3 were determined to be positive by RT-PCR, and 10 had anti-RVFV IgM. Among them, one was positive for both. Given that previous studies have reported the circulation of RVFV during inter-epidemic periods and the occurrence of outbreaks due to imported RVFV in Madagascar, our findings suggest the importance of strengthening RVF surveillance from a "One Health" perspective by conducting syndromic and risk-based surveillance at the national and regional levels.

Socio-ecological risk factors associated with human flea infestations of rural household in plague-endemic areas of Madagascar.

Plague is a flea-borne fatal disease caused by the bacterium Yersinia pestis, which persists in rural Madagascar. Although fleas parasitizing rats are considered the primary vectors of Y. pestis, the human flea, Pulex irritans, is abundant in human habitations in Madagascar, and has been found naturally infected by the plague bacterium during outbreaks. While P. irritans may therefore play a role in plague transmission if present in plague endemic areas, the factors associated with infestation and human exposure within such regions are little explored. To determine the socio-ecological risk factors associated with P. irritans infestation in rural households in plague-endemic areas of Madagascar, we used a mixed-methods approach, integrating results from P. irritans sampling, a household survey instrument, and an observational checklist. Using previously published vectorial capacity data, the minimal P. irritans index required for interhuman bubonic plague transmission was modeled to determine whether household infestations were enough to pose a plague transmission risk. Socio-ecological risk factors associated with a high P. irritans index were then identified for enrolled households using generalized linear models. Household flea abundance was also modeled using the same set of predictors. A high P. irritans index occurred in approximately one third of households and was primarily associated with having a traditional dirt floor covered with a plant fiber mat. Interventions targeting home improvement and livestock housing management may alleviate flea abundance and plague risk in rural villages experiencing high P. irritans infestation. As plague-control resources are limited in developing countries such as Madagascar, identifying the household parameters and human behaviors favoring flea abundance, such as those identified in this study, are key to developing preventive measures that can be implemented at the community level.

Noninvasive detection of Zika virus in mosquito excreta sampled from wild mosquito populations in French Guiana.

Arboviruses can be difficult to detect in the field due to relatively low prevalence in mosquito populations. The discovery that infected mosquitoes can release viruses in both their saliva and excreta gave rise to low-cost methods for the detection of arboviruses during entomological surveillance. We implemented both saliva and excreta-based entomological surveillance during the emergence of Zika virus (ZIKV) in French Guiana in 2016 by trapping mosquitoes around households of symptomatic cases with confirmed ZIKV infection. ZIKV was detected in mosquito excreta and not in mosquito saliva in 1 trap collection out of 85 (1.2%). One female Ae. aegypti L. (Diptera: Culicidae) was found with a ZIKV systemic infection in the corresponding trap. The lag time between symptom onset in a ZIKV-infected individual living near the trap site and ZIKV detection in this mosquito was 1 wk. These results highlight the potential of detection in excreta from trapped mosquitoes as a sensitive and cost-effective method to non invasively detect arbovirus circulation.

Surveillance of Fleas and Their Small Mammal Hosts for Plague Risks in Some Main Seaports of the Islands of the Southwestern Indian Ocean.

Since ancient times, seaports have been the hot spots for plague introduction into free countries. Infected ship rats reached new areas, and epizootics occurred prior to human infection via flea bites. Beginning in the 1920s/1930s, rodent and flea surveillance was carried out as part of plague hazard management in seaports of the world. Nowadays, such activity is not done regularly. In the southwestern Indian Ocean (SWIO) region, plague surveillance is of great importance given plague endemicity in Madagascar and thus the incurred risk for neighboring islands. This study reports animal-based surveillance aimed at identifying fleas and their small mammal hosts in SWIO seaports as well as Yersinia pestis detection. Small mammal trappings were performed in five main seaports of Madagascar (Toamasina and Mahajanga), Mauritius (Port Louis), and the Union of Comoros (Moroni and Mutsamudu). Mammals were euthanized and their fleas collected and morphologically identified before Y. pestis detection. In total, 145 mammals were trapped: the brown rat Rattus norvegicus (76.5%), the black rat Rattus rattus (8.3%), and the Asian house shrew Suncus murinus (15.2%). Fur brushing allowed collection of 1,596 fleas exclusively identified as Xenopsylla cheopis. All tested fleas were negative for Y. pestis DNA. This study shows that both well-known plague mammal hosts and flea vectors occur in SWIO seaports. It also highlights the necessity of carrying out regular animal-based surveillance for plague hazard management in this region.

Anopheles gambiae on remote islands in the Indian Ocean: origins and prospects for malaria elimination by genetic modification of extant populations.

The mosquito Anopheles gambiae s.s. is a primary malaria vector throughout sub-Saharan Africa including the islands of the Comoros archipelago (Anjouan, Grande Comore, Mayotte and Mohéli). These islands are located at the northern end of the Mozambique Channel in eastern Africa. Previous studies have shown a relatively high degree of genetic isolation between the Comoros islands and mainland populations of A. gambiae, but the origin of the island populations remains unclear. Here, we analyzed phylogenetic relationships among island and mainland populations using complete mitochondrial genome sequences of individual A. gambiae specimens. This work augments earlier studies based on analysis of the nuclear genome. We investigated the source population of A. gambiae for each island, estimated the number of introductions, when they occurred and explored evidence for contemporary gene flow between island and mainland populations. These studies are relevant to understanding historical patterns in the dispersal of this important malaria vector and provide information critical to assessing their potential for the exploration of genetic-based vector control methods to eliminate this disease. Phylogenetic analysis and haplotype networks were constructed from mitogenome sequences of 258 A. gambiae from the four islands. In addition, 112 individuals from seven countries across sub-Saharan Africa and Madagascar were included to identify potential source populations. Our results suggest that introduction events of A. gambiae into the Comoros archipelago were rare and recent events and support earlier claims that gene flow between the mainland and these islands is limited. This study is concordant with earlier work suggesting the suitability of these oceanic islands as appropriate sites for conducting field trial releases of genetically engineered mosquitoes (GEMs).

Comprehensive Characterization of Viral Diversity of Female Mosquitoes in Madagascar.

The diversity and circulation of arboviruses are not much studied in Madagascar. The fact is that arboviral emergences are rarely detected. The existing surveillance system primarily relies on serological detection and records only a few human infections annually. The city of Mahajanga, however, experienced a confirmed dengue fever epidemic in 2020 and 2021. This study aimed to characterize and analyze the virome of mosquitoes collected in Mahajanga, near patients with dengue-like syndromes to detect known and unknown viruses as well as investigate the factors contributing to the relative low circulation of arboviruses in the area. A total of 4280 mosquitoes representing at least 12 species from the Aedes, Anopheles, and Culex genera were collected during the dry and the rainy seasons from three sites, following an urbanization gradient. The virome analysis of 2192 female mosquitoes identified a diverse range of viral families and genera and revealed different patterns that are signatures of the influence of the mosquito genus or the season of collection on the composition and abundance of the virome. Despite the absence of known human or veterinary arboviruses, the identification and characterization of viral families, genera, and species in the mosquito virome contribute to our understanding of viral ecology and diversity within mosquito populations in Madagascar. This study serves as a foundation for ongoing surveillance efforts and provides a basis for the development of preventive strategies against various mosquito-borne viral diseases, including known arboviruses.

Knockdown resistance mutations are common and widely distributed in Xenopsylla cheopis fleas that transmit plague in Madagascar.

BACKGROUND: Plague, caused by the bacterium Yersinia pestis, remains an important disease in Madagascar, where the oriental rat flea, Xenopsylla cheopis, is a primary vector. To control fleas, synthetic pyrethroids (SPs) have been used for >20 years, resulting in resistance in many X. cheopis populations. The most common mechanisms of SP resistance are target site mutations in the voltage-gated sodium channel (VGSC) gene. METHODOLOGY/PRINCIPAL FINDINGS: We obtained 25 collections of X. cheopis from 22 locations across Madagascar and performed phenotypic tests to determine resistance to deltamethrin, permethrin, and/or dichlorodiphenyltrichloroethane (DDT). Most populations were resistant to all these insecticides. We sequenced a 535 bp segment of the VGSC gene and identified two different mutations encoding distinct substitutions at amino acid position 1014, which is associated with knockdown resistance (kdr) to SPs in insects. Kdr mutation L1014F occurred in all 25 collections; a rarer mutation, L1014H, was found in 12 collections. There was a significant positive relationship between the frequency of kdr alleles and the proportion of individuals surviving exposure to deltamethrin. Phylogenetic comparisons of 12 VGSC alleles in Madagascar suggested resistant alleles arose from susceptible lineages at least three times. Because genotype can reasonably predict resistance phenotype, we developed a TaqMan PCR assay for the rapid detection of kdr resistance alleles. CONCLUSIONS/SIGNIFICANCE: Our study provides new insights into VGSC mutations in Malagasy populations of X. cheopis and is the first to report a positive correlation between VGSC genotypes and SP resistance phenotypes in fleas. Widespread occurrence of these two SP resistance mutations in X. cheopis populations in Madagascar reduces the viability of these insecticides for flea control. However, the TaqMan assay described here facilitates rapid detection of kdr mutations to inform when use of these insecticides is still warranted to reduce transmission of plague.

Ribosomal RNA (rRNA) sequences from 33 globally distributed mosquito species for improved metagenomics and species identification.

Total RNA sequencing (RNA-seq) is an important tool in the study of mosquitoes and the RNA viruses they vector as it allows assessment of both host and viral RNA in specimens. However, there are two main constraints. First, as with many other species, abundant mosquito ribosomal RNA (rRNA) serves as the predominant template from which sequences are generated, meaning that the desired host and viral templates are sequenced far less. Second, mosquito specimens captured in the field must be correctly identified, in some cases to the sub-species level. Here, we generate mosquito rRNA datasets which will substantially mitigate both of these problems. We describe a strategy to assemble novel rRNA sequences from mosquito specimens and produce an unprecedented dataset of 234 full-length 28S and 18S rRNA sequences of 33 medically important species from countries with known histories of mosquito-borne virus circulation (Cambodia, the Central African Republic, Madagascar, and French Guiana). These sequences will allow both physical and computational removal of rRNA from specimens during RNA-seq protocols. We also assess the utility of rRNA sequences for molecular taxonomy and compare phylogenies constructed using rRNA sequences versus those created using the gold standard for molecular species identification of specimens-the mitochondrial cytochrome c oxidase I (COI) gene. We find that rRNA- and COI-derived phylogenetic trees are incongruent and that 28S and concatenated 28S+18S rRNA phylogenies reflect evolutionary relationships that are more aligned with contemporary mosquito systematics. This significant expansion to the current rRNA reference library for mosquitoes will improve mosquito RNA-seq metagenomics by permitting the optimization of species-specific rRNA depletion protocols for a broader range of species and streamlining species identification by rRNA sequence and phylogenetics.

Biodiversity and vector-borne diseases: Host dilution and vector amplification occur simultaneously for Amazonian leishmaniases.

Changes in biodiversity may impact infectious disease transmission through multiple mechanisms. We explored the impact of biodiversity changes on the transmission of Amazonian leishmaniases, a group of wild zoonoses transmitted by phlebotomine sand flies (Psychodidae), which represent an important health burden in a region where biodiversity is both rich and threatened. Using molecular analyses of sand fly pools and blood-fed dipterans, we characterized the disease system in forest sites in French Guiana undergoing different levels of human-induced disturbance. We show that the prevalence of Leishmania parasites in sand flies correlates positively with the relative abundance of mammal species known as Leishmania reservoirs. In addition, Leishmania reservoirs tend to dominate in less diverse mammal communities, in accordance with the dilution effect hypothesis. This results in a negative relationship between Leishmania prevalence and mammal diversity. On the other hand, higher mammal diversity is associated with higher sand fly density, possibly because more diverse mammal communities harbor higher biomass and more abundant feeding resources for sand flies, although more research is needed to identify the factors that shape sand fly communities. As a consequence of these antagonistic effects, decreased mammal diversity comes with an increase of parasite prevalence in sand flies, but has no detectable impact on the density of infected sand flies. These results represent additional evidence that biodiversity changes may simultaneously dilute and amplify vector-borne disease transmission through different mechanisms that need to be better understood before drawing generalities on the biodiversity-disease relationship.

Variation in Pyrethroid Resistance Phenotypes in Anopheles darlingi in an Area with Residual Malaria Transmission: Warning of Suspected Resistance in French Guiana.

Anopheles darlingi is the main vector of malaria in South America. In French Guiana, malaria transmission occurs inland and along the rivers with a regular reemergence in the lower Oyapock area. Control against malaria vectors includes indoor residual spraying of deltamethrin and the distribution of long-lasting impregnated bednets. In this context, the level of resistance to pyrethroids was monitored for 4 years using CDC bottle tests in An. darlingi populations. A loss of susceptibility to pyrethroids was recorded with 30-minute knock-down measured as low as 81%. However, no pyrethroid molecular resistance was found by sequencing a 170 base pair fragment of the S6 segment of domain II of the voltage-gated sodium channel gene. Fluctuation of resistance phenotypes may be influenced by the reintroduction of susceptible alleles from sylvatic populations or by other mechanisms of metabolic resistance.

MALDI-TOF MS: An effective tool for a global surveillance of dengue vector species.

Dengue, Zika and chikungunya viruses cause significant human public health burdens in the world. These arboviruses are transmitted by vector mosquito species notably Aedes aegypti and Aedes albopictus. In the Pacific region, more vector species of arboviruses belonging to the Scutellaris Group are present. Due to the expansion of human travel and international trade, the threat of their dispersal in other world regions is on the rise. Strengthening of entomological surveillance ensuring rapid detection of introduced vector species is therefore required in order to avoid their establishment and the risk of arbovirus outbreaks. This surveillance relies on accurate species identification. The aim of this study was to assess the use of the Matrix-Assisted Laser Desorption Ionization Time-Of-Flight Mass Spectrometry (MALDI-TOF MS) as a tool for an international identification and surveillance of these mosquito vectors of arboviruses. Field-mosquitoes belonging to 8 species (Ae. aegypti, Ae. albopictus, Aedes polynesiensis, Aedes scutellaris, Aedes pseudoscutellaris, Aedes malayensis, Aedes futunae and Culex quinquefasciatus) from 6 countries in the Pacific, Asian and Madagascar, were included in this study. Analysis provided evidence that a MALDI-TOF database created using mosquitoes from the Pacific region allowed suitable identification of mosquito species from the other regions. This technic was as efficient as the DNA sequencing method in identifying mosquito species. Indeed, with the exception of two Ae. pseudoscutellaris, an exact species identification was obtained for all individual mosquitoes. These findings highlight that the MALDI-TOF MS is a promising tool that could be used for a global comprehensive arbovirus vector surveillance.

Interactions between vector competence to chikungunya virus and resistance to deltamethrin in Aedes aegypti laboratory lines?

The urban mosquito species Aedes aegypti is the main vector of arboviruses worldwide. Mosquito control with insecticides is the most prevalent method for preventing transmission in the absence of effective vaccines and available treatments; however, the extensive use of insecticides has led to the development of resistance in mosquito populations throughout the world, and the number of epidemics caused by arboviruses has increased. Three mosquito lines with different resistance profiles to deltamethrin were isolated in French Guiana, including one with the I1016 knock-down resistant allele. Significant differences were observed in the cumulative proportion of mosquitoes with a disseminated chikungunya virus infection over time across these lines. In addition, some genes related to resistance (CYP6BB2, CYP6N12, GST2, trypsin) were variably overexpressed in the midgut at 7 days after an infectious bloodmeal in these three lines. Our work shows that vector competence for chikungunya virus varied between Ae. aegypti laboratory lines with different deltamethrin resistance profiles. More accurate verification of the functional association between insecticide resistance and vector competence remains to be demonstrated.

Tracking of Mammals and Their Fleas for Plague Surveillance in Madagascar, 2018-2019.

Plague, a zoonotic disease caused by the bacterium Yersinia pestis, remains a major public health threat in Madagascar. To better understand the risk of transmission to humans and to guide targeted plague prevention and control measures, a survey of Y. pestis infection and exposure in mammals and their fleas was implemented. Small mammals were captured in five districts of Madagascar ranging in levels of plague endemicity, as measured by notified cases, from none to active foci. Blood and spleen samples and fleas were collected from small mammals for the detection of anti-Y. pestis F1 antibodies by ELISA, F1 antigens by rapid diagnostic tests, and pla, caf1, and inv genes by polymerase chain reaction. Some rodent fleas were kept alive and reared in the insectary to assess susceptibility to insecticides. Blood was also collected from 15 dogs and tested for anti-F1 antibodies. A total of 557 spleens, 484 sera, and 1,539 fleas were collected from 557 rodents and shrews. Nineteen (3.4%) spleens were positive for F1 antigen, most from Toamasina (N = 13), a historical plague focus. One dog was also found seropositive in Toamasina. Twenty-two (4.5%) serologic specimens from small mammals were positive for anti-F1 antibodies. The flea index was highest in the city of Antananarivo (8.8). No flea was positive for Y. pestis DNA. Flea populations exhibited resistance to various insecticides weakening the efficacy of vector control. This study highlights the potential use of animal-based surveillance to identify the risk of plague transmission in endemic and nonendemic foci for targeted prevention and control.

A Francisella tularensis-Like Bacterium in Tropical Bed Bugs from Madagascar.

The genus Francisella includes several highly virulent human pathogens and some tick endosymbionts. Francisella infections are acquired by humans through contact with vertebrate animal reservoirs or contaminated water or dust. The species Francisella tularensis can also be transmitted by arthropods including ticks, mosquitoes, and flies. For the first time, we describe the molecular detection of an F. tularensis-like bacterium in bed bugs from samples collected in rural Madagascar. This finding suggests a potential involvement of bed bugs in the ecology of Francisella. The role of bed bugs as possible hosts, reservoirs, or vectors of Francisella spp. should be further investigated.

Illustrated Morphological Keys for Fleas (Siphonaptera) in Madagascar.

Madagascar has an important diversity of fleas (Siphonaptera), which almost species do not exist elsewhere. Scientists have provided huge efforts to improve knowledge on Malagasy fleas since the middle of 1900s particularly by investigating topics such as taxonomy, systematics, biogeography, and flea vector role. Since then, new species discovery has increased and currently, 48 species are recorded which the majority is endemic. Therefore, it becomes necessary to have updated keys to identify species membership. This paper presents morphological-based keys to identify at genus and species levels adult fleas reported as occurring in Madagascar. Illustrations are proposed to make easier the observation of morphological criteria, which may be tricky for sibling species.

The Culex Mosquitoes (Diptera: Culicidae) of French Guiana: A Comprehensive Review With the Description of Three New Species.

The taxonomically intricate genus Culex Linnaeus includes numerous known vector species of parasites and viruses to humans. The aim of this article is to comprehensively review the Culex species which occur in French Guiana to provide a stronger taxonomic foundation for future studies on this genus in South America. The occurrence of Culex species was investigated in light of current taxonomic knowledge through an extensive examination of voucher specimens deposited in the entomological collections of four French depositories and additional specimens recently collected at various localities in French Guiana. Based on this review, 104 Culex species classified in eight subgenera are confirmed to occur in French Guiana. Compared to the most recent checklist, 18 species are added, and 10 species excluded, resulting in a total number of 242 valid mosquito species known to occur in French Guiana. Three nominal species are synonymized, three others are newly described, and a last one is transferred to another informal infrasubgeneric group. Overall, this review also highlights the limits of the use of only bibliographic data when dealing with taxonomically complex groups of insects.

Study Protocol: A Cross-Sectional Examination of Socio-Demographic and Ecological Determinants of Nutrition and Disease Across Madagascar.

Madagascar has experienced significant environmental change since 1960, particularly through forest clearing for agricultural expansion. Climatic patterns are undergoing change in Madagascar as well, with increasing temperatures, droughts, and cyclonic activity. The impact of these environmental and climatic changes will pose threats to food availability, income generation, and local ecosystems, with significant potential effects on the spatial and temporal distribution of disease burden. This study seeks to describe the health status of a large sample of geographically and socially diverse Malagasy communities through multiple clinical measurements, detailed social surveys, and paired data on regional variation in local ecologies. With an increased understanding of the current patterns of variation in human health and nutrition, future studies will be better able to identify associations with climate and anticipate and mitigate the burdens expected from larger, longer-term changes. Our mixed-method approach included an observational cross-sectional study. Research subjects were men, women, and children from 1,125 households evenly distributed across 24 communities in four ecologically and socio-demographically distinct regions of Madagascar. For these 1,125 households, all persons of both sexes and all ages therein (for a total of 6,292 individuals) were recruited into the research study and a total of 5,882 individuals were enrolled. Through repeated social survey recalls and focus group meetings, we obtained social and demographic data, including broad categories of seasonal movements, and characterized the fluctuation of income generation, food production and dietary consumption. Through collection of clinical and biological samples for both point-of-care diagnoses and laboratory analyses, we obtained detailed occurrence (and importantly co-occurrence) data on micronutrient nutritional, infectious disease, and non-communicable disease status. Our research highlights the highly variable social, cultural, and environmental contexts of health conditions in Madagascar, and the tremendous inter-regional, inter-community, and intra-community variation in nutritional and disease status. More than 30% of the surveyed population was afflicted by anemia and 14% of the population had a current malaria infection. This type of rich metadata associated with a suite of biological samples and nutritional and disease outcome data should allow disentangling some of the underlying drivers of ill health across the changing landscapes of Madagascar.

Discrimination of Uranotaenia species (Diptera: Culicidae) from Madagascar based on morphology and wing morphometric traits.

The genus Uranotaenia (Diptera: Culicidae) has been well documented in Madagascar where it includes 73 species, 89.4% being endemic. However, one problem is that most species are morphologically similar in the adult stage. Here, 713 Uranotaenia specimens collected in the tropical forests of Anorana and Maromizaha between 2008 and 2014 were examined. Using the dichotomous keys for the Uranotaenia fauna of Madagascar published in 2004, three species were identified: Uranotaenia neireti (220), Ur. alboabdominalis (110) and Ur. mayottensis (28). The other specimens (355) were not identifiable and were classified as Uranotaenia sp1. Using wing morphometry, the four taxa were classified into four morphogroups. Within the Uranotaenia sp1 group, specimens from the Anorana forest and those from the Maromizaha forest overlapped. This result suggests that wing morphometric traits could be a good marker to distinguish Uranotaenia species in Madagascar.

Variation in Anopheles distribution and predictors of malaria infection risk across regions of Madagascar.

BACKGROUND: Deforestation and land use change is widespread in Madagascar, altering local ecosystems and creating opportunities for disease vectors, such as the Anopheles mosquito, to proliferate and more easily reach vulnerable, rural populations. Knowledge of risk factors associated with malaria infections is growing globally, but these associations remain understudied across Madagascar's diverse ecosystems experiencing rapid environmental change. This study aims to uncover socioeconomic, demographic, and ecological risk factors for malaria infection across regions through analysis of a large, cross-sectional dataset. METHODS: The objectives were to assess (1) the ecological correlates of malaria vector breeding through larval surveys, and (2) the socioeconomic, demographic, and ecological risk factors for malaria infection in four ecologically distinct regions of rural Madagascar. Risk factors were determined using multilevel models for the four regions included in the study. RESULTS: The presence of aquatic agriculture (both within and surrounding communities) is the strongest predictive factor of habitats containing Anopheles larvae across all regions. Ecological and socioeconomic risk factors for malaria infection vary dramatically across study regions and range in their complexity. CONCLUSIONS: Risk factors for malaria transmission differ dramatically across regions of Madagascar. These results may help stratifying current malaria control efforts in Madagascar beyond the scope of existing interventions.