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The International Centers of Excellence for Malaria Research (ICEMR) were established by the National Institute of Allergy and Infectious Diseases more than a decade ago to provide multidisciplinary research support to malaria control programs worldwide, operating in endemic areas and contributing technology, expertise, and ultimately policy guidance for malaria control and elimination. The Southern and Central Africa ICEMR has conducted research across three main sites in Zambia and Zimbabwe that differ in ecology, entomology, transmission intensity, and control strategies. Scientific findings led to new policies and action by the national malaria control programs and their partners in the selection of methods, materials, timing, and locations of case management and vector control. Malaria risk maps and predictive models of case detection furnished by the ICEMR informed malaria elimination programming in southern Zambia, and time series analyses of entomological and parasitological data motivated several major changes to indoor residual spray campaigns in northern Zambia. Along the Zimbabwe-Mozambique border, temporal and geospatial data are currently informing investigations into a recent resurgence of malaria. Other ICEMR findings pertaining to parasite and mosquito genetics, human behavior, and clinical epidemiology have similarly yielded immediate and long-term policy implications at each of the sites, often with generalizable conclusions. The ICEMR programs thereby provide rigorous scientific investigations and analyses to national control and elimination programs, without which the impediments to malaria control and their potential solutions would remain understudied.

For a decade, the Southern and Central Africa International Center of Excellence for Malaria Research has operated with local partners across study sites in Zambia and Zimbabwe that range from hypo- to holoendemic and vary ecologically and entomologically. The burden of malaria and the impact of control measures were assessed in longitudinal cohorts, cross-sectional surveys, passive and reactive case detection, and other observational designs that incorporated multidisciplinary scientific approaches: classical epidemiology, geospatial science, serosurveillance, parasite and mosquito genetics, and vector bionomics. Findings to date have helped elaborate the patterns and possible causes of sustained low-to-moderate transmission in southern Zambia and eastern Zimbabwe and recalcitrant high transmission and fatality in northern Zambia. Cryptic and novel mosquito vectors, asymptomatic parasite reservoirs in older children, residual parasitemia and gametocytemia after treatment, indoor residual spraying timed dyssynchronously to vector abundance, and stockouts of essential malaria commodities, all in the context of intractable rural poverty, appear to explain the persistent malaria burden despite current interventions. Ongoing studies of high-resolution transmission chains, parasite population structures, long-term malaria periodicity, and molecular entomology are further helping to lay new avenues for malaria control in southern and central Africa and similar settings.

Malaria transmission has declined substantially in Southern Province, Zambia, which is considered a low-transmission setting. The Zambian government introduced a reactive test-and-treat strategy to identify active zones of transmission and treat parasitemic residents. This study was conducted in the Choma District, Southern Province, Zambia, concurrently with an evaluation of this strategy to identify vectors responsible for sustaining transmission, and to identify entomological, spatial, and ecological risk factors associated with increased densities of mosquitoes. Anophelines were collected with CDC light traps indoors and near animal pens in index cases and neighboring households. Outdoor collections captured significantly more anophelines than indoor traps, and 10 different anopheline species were identified. Four species (Anopheles arabiensis, An. rufipes, An. squamosus, and An. coustani) were positive for Plasmodium falciparum circumsporozoite protein by ELISA, and 61% of these 26 anophelines were captured outdoors. Bloodmeal assays confirm plasticity in An. arabiensis foraging, feeding both on humans and animals, whereas An. rufipes, An. squamosus, and An. coustani were largely zoophilic and exophilic. Linear regression of count data for indoor traps revealed that households with at least one parasitemic resident by polymerase chain reaction testing was associated with higher female anopheline counts. This suggests that targeting households with parasitemic individuals for vector interventions may reduce indoor anopheline populations. However, many vectors species responsible for transmission may not be affected by indoor interventions because they are primarily exophilic and forage opportunistically. These data underscore the necessity for further evaluation of vector surveillance and control tools that are effective outdoors, in conjunction with current indoor-based interventions.

Despite dramatic reductions in malaria cases in the catchment area of Macha Hospital, Choma District, Southern Province in Zambia, prevalence has remained near 1-2% by RDT for the past several years. To investigate residual malaria transmission in the area, this study focuses on the relative abundance, foraging behavior, and phylogenetic relationships of Anopheles squamosus specimens. In 2011, higher than expected rates of anthropophily were observed among "zoophilic" An. squamosus , a species that had sporadically been found to contain Plasmodium falciparum sporozoites. The importance of An. squamosus in the region was reaffirmed in 2016 when P. falciparum sporozoites were detected in numerous An. squamosus specimens. This study analyzed Centers for Disease Control (CDC) light trap collections of adult mosquitoes from two collection schemes: one performed as part of a reactive-test-and-treat program and the second performed along a geographical transect. Morphological identification, molecular verification of anopheline species, and blood meal source were determined on individual samples. Data from these collections supported earlier studies demonstrating An. squamosus to be primarily exophagic and zoophilic, allowing them to evade current control measures. The phylogenetic relationships generated from the specimens in this study illustrate the existence of well supported clade structure among An. squamosus specimens, which further emphasizes the importance of molecular identification of vectors. The primarily exophagic behavior of An. squamosus in these collections also highlights that indoor vector control strategies will not be sufficient for elimination of malaria in southern Zambia.

Residual vector populations that do not come in contact with the most frequently utilized indoor-directed interventions present major challenges to global malaria eradication. Many of these residual populations are mosquito species about which little is known. As part of a study to assess the threat of outdoor exposure to malaria mosquitoes within the Southern and Central Africa International Centers of Excellence for Malaria Research, foraging female anophelines were collected outside households in Nchelenge District, northern Zambia. These anophelines proved to be more diverse than had previously been reported in the area. In order to further characterize the anopheline species, sequencing and phylogenetic approaches were utilized. Anopheline mosquitoes were collected from outdoor light traps, morphologically identified, and sent to Johns Hopkins Bloomberg School of Public Health for sequencing. Sanger sequencing from 115 field-derived samples yielded mitochondrial COI sequences, which were aligned with a homologous 488 bp gene segment from known anophelines (n = 140) retrieved from NCBI. Nuclear ITS2 sequences (n = 57) for at least one individual from each unique COI clade were generated and compared against NCBI's nucleotide BLAST database to provide additional evidence for taxonomical identity and structure. Molecular and morphological data were combined for assignment of species or higher taxonomy. Twelve phylogenetic groups were characterized from the COI and ITS2 sequence data, including the primary vector species Anopheles funestus s.s. and An. gambiae s.s. An unexpectedly large proportion of the field collections were identified as An. coustani and An . sp. 6 . Six phylogenetic groups remain unidentified to species-level. Outdoor collections of anopheline mosquitoes in areas frequented by people in Nchelenge, northern Zambia, proved to be extremely diverse. Morphological misidentification and underrepresentation of some anopheline species in sequence databases confound efforts to confirm identity of potential malaria vector species. The large number of unidentified anophelines could compromise the malaria vector surveillance and malaria control efforts not only in northern Zambia but other places where surveillance and control are focused on indoor-foraging and resting anophelines. Therefore, it is critical to continue development of methodologies that allow better identification of these populations and revisiting and cleaning current genomic databases., Competing Interests: Conflict of Interest: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Background: The emergence of mosquitoes that can avoid indoor-deployed interventions, such as treated bed nets and indoor residual spraying, threatens the mainstay of malaria control in Zambia. Furthermore, the requirement for high coverage of these tools poses operational challenges. Spatial repellents are being assessed to supplement these vector control tools, but limitations exist in the residual effect of the repellent and the need for external power or heat for diffusion of the volatiles., Methods: A semi-field evaluation of a novel controlled release spatial repellent device (CRD) was conducted in Macha, Zambia. These devices emanate metofluthrin with no need for external power. Devices were deployed in huts within the semi-field system (SFS). Female Anopheles gambiae sensu stricto released within the SFS were trapped overnight by light traps and collected by aspiration the next morning inside and outside of huts to determine the extent of mosquito repellency and the impact on host-seeking and survival. Experiments studied the impact of number of devices as well as the presence of hut occupants. The study was complemented with numerical methods based on computational fluid dynamics to simulate spatial distribution of metofluthrin., Results: Presence of CRDs was associated with significant reductions in indoor counts of mosquitoes, regardless of whether huts were occupied or not. Repellency ranged from 15 to 60% compared to huts with no devices. Reducing the number of devices from 16 to 4 had little impact on repellency. When huts were occupied, indoor mosquito host-seeking was higher in the presence of CRDs, whilst survival was significantly reduced., Conclusions: This study demonstrated that deployment of as few as four CRDs within a hut was associated with reduced indoor mosquito densities. As would be expected, presence of occupants within huts, resulted in greater indoor catches (both with and without devices). The increased indoor mosquito host-seeking and mortality in huts when devices were present may be explained by the excito-repellency activity of metofluthrin. These semi-field experiments provide preliminary data on the utility of CRD spatial repellents to reduce indoor densities of An. gambiae mosquitoes. Studies will further investigate the impact of CRDs on mosquito behaviour as well as epidemiological protective efficacy.

Southern Zambia is the focus of strategies to create malaria-free zones. Interventions being rolled out include test and treat strategies and distribution of insecticide-treated bed nets that target vectors that host-seek indoors and late at night. In Macha, Choma District, collections of mosquitoes were made outdoors using barrier screens within homesteads or UV bulb light traps set next to goats, cattle, or chickens during the rainy season of 2015. Anopheline mosquitoes were identified to species using molecular methods and Plasmodium falciparum infectivity was determined by ELISA and real-time qPCR methods. More than 40% of specimens caught were identified as Anopheles squamosus Theobald, 1901 of which six were found harboring malaria parasites. A single sample, morphologically identified as Anopheles coustani Laveran, 1900, was also found to be infectious. All seven specimens were caught outdoors next to goat pens. Parasite-positive specimens as well as a subset of An. squamosus specimens from either the same study or archive collections from the same area underwent sequencing of the mitochondrial cytochrome oxidase subunit I gene. Maximum parsimony trees constructed from the aligned sequences indicated presence of at least two clades of An. squamosus with infectious specimens falling in each clade. The single infectious specimen identified morphologically as An. coustani could not be matched to reference sequences. This is the first report from Zambia of infections in An. squamosus, a species which is described in literature to display exophagic traits. The bionomic characteristics of this species needs to be studied further to fully evaluate the implications for indoor-targeted vector control., (© The Authors 2016. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)