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Antimalarial medications are recommended for chemoprevention as part of malaria control programs to decrease the morbidity and mortality related to more than 200 million infections each year. We sought to evaluate patient and provider acceptability of malaria chemoprevention in a long-acting formulation. We administered questionnaires to patients and providers in malaria endemic districts in Kenya and Zambia. Questions explored preferences and concerns around long-acting antimalarial formulations compared with oral formulations. We recruited 202 patient respondents (Kenya, n = 102; Zambia, n = 100) and 215 provider respondents (Kenya, n = 105; Zambia, n = 110). Long-acting injection was preferred to oral pills, whereas oral pills were preferred to implant or transdermal administration by patient respondents. Of 202 patient respondents, 80% indicated that they 'definitely would try' malaria chemoprevention offered by injection instead of oral pills. Of parents or guardians, 84% of 113 responded that they 'definitely would' have their child age < 12 years and 90% of 88 'definitely would' have their child ≥12 years receive an injection for malaria prevention. Provider respondents indicated that they would be more likely to prescribe a long-acting injectable product compared with an oral product for malaria chemoprevention in adults (70%), adolescents ages 12 years and older (67%), and children <12 years (81%). Potential for prolonged adverse effects with long-acting products was the highest concern for patient respondents, while higher medication-related cost was cited as the most concerning barrier to implementation by providers. Overall, these findings indicate enthusiasm for the development of long-acting injectable antimalarials to provide individual delivery method options across age groups.

Background: Understanding temporal and spatial dynamics of malaria transmission will help to inform effective interventions and strategies in regions approaching elimination. Parasite genomics are increasingly used to monitor epidemiologic trends, including assessing residual transmission across seasons and importation of malaria into these regions., Methods: In a low and seasonal transmission setting of southern Zambia, a total of 441 Plasmodium falciparum samples collected from 8 neighbouring health centres between 2012 and 2018 were genotyped using molecular inversion probes (MIPs n = 1793) targeting a total of 1832 neutral and geographically informative SNPs distributed across the parasite genome. After filtering for quality and missingness, 302 samples and 1410 SNPs were retained and used for downstream population genomic analyses., Results: The analyses revealed most (67%, n = 202) infections harboured one clone (monogenomic) with some variation at local level suggesting low, but heterogenous malaria transmission. Relatedness identity-by-descent (IBD) analysis revealed variable distribution of IBD segments across the genome and 6% of pairs were highly-related (IBD ≥ 0.25). Some of the highly-related parasite populations persisted across multiple seasons, suggesting that persistence of malaria in this low-transmission region is fueled by parasites "seeding" across the dry season. For recent years, clusters of clonal parasites were identified that were dissimilar to the general parasite population, suggesting parasite populations were increasingly fragmented at small spatial scales due to intensified control efforts. Clustering analysis using PCA and t-SNE showed a lack of substantial parasite population structure., Conclusion: Leveraging both genomic and epidemiological data provided comprehensive picture of fluctuations in parasite populations in this pre-elimination setting of southern Zambia over 7 years., (© 2023. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Despite efforts to minimize the impacts of malaria and reduce the number of primary vectors, malaria has yet to be eliminated in Zambia. Understudied vector species may perpetuate malaria transmission in pre-elimination settings. Anopheles squamosus is one of the most abundantly caught mosquito species in southern Zambia and has previously been found with Plasmodium falciparum sporozoites, a causal agent of human malaria. This species may be a critical vector of malaria transmission, however, there is a lack of genetic information available for An. squamosus. We report the first genome data and the first complete mitogenome (Mt) sequence of An. squamosus. The sequence was extracted from one individual mosquito from the Chidakwa area in Macha, Zambia. The raw reads were obtained using Illumina Novaseq 6000 and assembled through NOVOplasty alignment with related species. The length of the An. squamosus Mt was 15,351 bp, with 77.9 % AT content. The closest match to the whole mitochondrial genome in the phylogenetic tree is the African malaria mosquito, Anopheles gambiae. Its genome data is available through National Center for Biotechnology Information (NCBI) Sequencing Reads Archive (SRA) with accession number SRR22114392. The mitochondrial genome was deposited in NCBI GenBank with the accession number OP776919. The ITS2 containing contig sequence was deposited in GenBank with the accession number OQ241725. Mitogenome annotation and a phylogenetic tree with related Anopheles mosquito species are provided., Competing Interests: No competing interests were disclosed., (Copyright: © 2023 Nguyen VT et al.)

Background: Nchelenge District in northern Zambia suffers from holoendemic malaria transmission despite a decade of yearly indoor residual spraying (IRS) and insecticide-treated net (ITN) distributions. One hypothesis for this lack of impact is that some vectors in the area may forage in the early evening or outdoors. Anopheles gibbinsi specimens were identified in early evening mosquito collections performed in this study area, and further insight was gleaned into this taxon, including characterizing its genetic identity, feeding preferences, and potential role as a malaria vector., Methods: Mosquitoes were collected in July and August 2019 by CDC light traps in Nchelenge District in indoor sitting rooms, outdoor gathering spaces, and animal pens from 16:00-22:00. Host detection by PCR, COI and ITS2 PCR, and circumsporozoite (CSP) ELISA were performed on all samples morphologically identified as An. gibbinsi, and a subset of specimens were selected for COI and ITS2 sequencing. To determine risk factors for increased abundance of An. gibbinsi, a negative binomial generalized linear mixed-effects model was performed with household-level variables of interest., Results: Comparison of COI and ITS2 An. gibbinsi reference sequences to the NCBI database revealed > 99% identity to "Anopheles sp. 6" from Kenya. More than 97% of specimens were morphologically and molecularly consistent with An. gibbinsi. Specimens were primarily collected in animal pen traps (59.2%), followed by traps outdoors near where humans gather (24.3%), and traps set indoors (16.5%). Host DNA detection revealed a high propensity for goats, but 5% of specimens with detected host DNA had fed on humans. No specimens were positive for Plasmodium falciparum sporozoites. Animal pens and inland households > 3 km from Lake Mweru were both associated with increased An. gibbinsi abundance., Conclusions: This is the first report of An. gibbinsi in Nchelenge District, Zambia. This study provided a species identity for unknown "An. sp. 6" in the NCBI database, which has been implicated in malaria transmission in Kenya. Composite data suggest that this species is largely zoophilic and exophilic, but comes into contact with humans and the malaria parasites they carry. This species should continue to be monitored in Zambia and neighbouring countries as a potential malaria vector., (© 2022. The Author(s).)

Background: Although vector-borne zoonotic diseases are a major public health threat globally, they are usually neglected, especially among resource-constrained countries, including those in sub-Saharan Africa. This scoping review examined the current knowledge and identified research gaps of vector-borne zoonotic pathogens in Zambia., Methods and Findings: Major scientific databases (Web of Science, PubMed, Scopus, Google Scholar, CABI, Scientific Information Database (SID)) were searched for articles describing vector-borne (mosquitoes, ticks, fleas and tsetse flies) zoonotic pathogens in Zambia. Several mosquito-borne arboviruses have been reported including Yellow fever, Ntaya, Mayaro, Dengue, Zika, West Nile, Chikungunya, Sindbis, and Rift Valley fever viruses. Flea-borne zoonotic pathogens reported include Yersinia pestis and Rickettsia felis. Trypanosoma sp. was the only tsetse fly-borne pathogen identified. Further, tick-borne zoonotic pathogens reported included Crimean-Congo Haemorrhagic fever virus, Rickettsia sp., Anaplasma sp., Ehrlichia sp., Borrelia sp., and Coxiella burnetii., Conclusions: This study revealed the presence of many vector-borne zoonotic pathogens circulating in vectors and animals in Zambia. Though reports of human clinical cases were limited, several serological studies provided considerable evidence of zoonotic transmission of vector-borne pathogens in humans. However, the disease burden in humans attributable to vector-borne zoonotic infections could not be ascertained from the available reports and this precludes the formulation of national policies that could help in the control and mitigation of the impact of these diseases in Zambia. Therefore, there is an urgent need to scale-up "One Health" research in emerging and re-emerging infectious diseases to enable the country to prepare for future epidemics, including pandemics., Competing Interests: The authors have declared that no competing interests exist.

The global malaria burden has decreased substantially, but gains have been uneven both within and between countries. In Zambia, the malaria burden remains high in northern and eastern regions of the country. To effectively reduce malaria transmission in these areas, evidence-based intervention strategies are needed. Zambia's National Malaria Control Centre conducted targeted indoor residual spraying (IRS) in 40 high-burden districts from 2014 to 2016 using the novel organophosphate insecticide pirimiphos-methyl. The Southern and Central Africa International Centers of Excellence for Malaria Research conducted an evaluation of the impact of the IRS campaign on household vector abundance in Nchelenge District, Luapula Province. From April 2012 to July 2017, field teams conducted indoor overnight vector collections from 25 to 30 households per month using Centers for Disease Control light traps. Changes in indoor anopheline counts before versus after IRS were assessed by species using negative binomial regression models with robust standard errors, controlling for geographic and climatological covariates. Counts of Anopheles funestus declined by approximately 50% in the study area and within areas targeted for IRS, and counts of Anopheles gambiae declined by approximately 40%. Within targeted areas, An. funestus counts declined more in sprayed households than in unsprayed households; however, this relationship was not observed for An. gambiae. The moderate decrease in indoor vector abundance indicates that IRS with pirimiphos-methyl is an effective vector control measure, but a more comprehensive package of interventions is needed with sufficient coverage to effectively reduce the malaria burden in this setting.

Artemether-lumefantrine (AL) is a first-line agent for uncomplicated malaria caused by Plasmodium falciparum . The WHO recommends periodic therapeutic efficacy studies of antimalarial drugs for the detection of malaria parasite drug resistance and to inform national malaria treatment policies. We conducted a therapeutic efficacy study of AL in a high malaria transmission region of northern Zambia from December 2014 to July 2015. One hundred children of ages 6 to 59 months presenting to a rural health clinic with uncomplicated falciparum malaria were admitted for treatment with AL (standard 6-dose regimen) and followed weekly for 5 weeks. Parasite counts were taken every 6 hours during treatment to assess parasite clearance. Recurrent episodes during follow-up ( n = 14) were genotyped to distinguish recrudescence from reinfection and to identify drug resistance single nucleotide polymorphisms (SNPs) and multidrug resistance protein 1 ( mdr1 ) copy number variation. Day 7 lumefantrine concentrations were measured for correspondence with posttreatment reinfection. All children who completed the parasite clearance portion of the study ( n = 94) were microscopy-negative by 72 hours. The median parasite elimination half-life was 2.7 hours (interquartile range: 2.1-3.3). Genotype-corrected therapeutic efficacy was 98.8% (95% CI: 97.6-100). Purported artemisinin and lumefantrine drug resistance SNPs in atp6 , 3D7&#95;1451200 , and mdr1 were detected but did not correlate with parasite recurrence, nor did day 7 lumefantrine concentrations. In summary, AL was highly effective for the treatment of uncomplicated falciparum malaria in northern Zambia during the study period. The high incidence of recurrent parasitemia was consistent with reinfection due to high, perennial malaria transmission.

Despite ongoing malaria control efforts implemented throughout sub-Saharan Africa, malaria remains an enormous public health concern. Current interventions such as indoor residual spraying with insecticides and use of insecticide-treated bed nets are aimed at targeting the key malaria vectors that are primarily endophagic and endophilic. Anopheles coustani s.l., an understudied vector of malaria, is a species previously thought to exhibit mostly zoophilic behavior. Like many of these understudied species, An. coustani has greater anthropophilic tendencies than previously appreciated, is often both endophagic and exophagic, and carries Plasmodium falciparum sporozoites. The aim of this study was to explore genetic variation of An. coustani mosquitoes and the potential of this species to contribute to malaria parasite transmission in high transmission settings in Zambia and the Democratic Republic of the Congo (DRC). Morphologically identified An. coustani specimens that were trapped outdoors in these study sites were analyzed by PCR and sequencing for species identification and bloodmeal sources, and malaria parasite infection was determined by ELISA and qPCR. Fifty An. coustani s.s. specimens were confirmed by analysis of mitochondrial DNA cytochrome c oxidase subunit I (COI) and ribosomal internal transcribed spacer region 2 (ITS2). Maximum likelihood phylogenetic analysis of COI and ITS2 sequences revealed two distinct phylogenetic groups within this relatively small regional collection. Our findings indicate that both An. coustani groups have anthropophilic and exophagic habits and come into frequent contact with P. falciparum, suggesting that this potential alternative malaria vector might elude current vector control measures in northern Zambia and southern DRC., (© The Author(s) 2020. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Although transmission of malaria and other mosquito-borne diseases is geographically heterogeneous, in sub-Saharan Africa risk maps are rarely used to determine which communities receive vector control interventions. We compared outcomes in areas receiving different indoor residual spray (IRS) strategies in Eastern Province, Zambia: (1) concentrating IRS interventions within a geographical area, (2) prioritizing communities to receive IRS based on predicted probabilities of Anopheles funestus, and (3) prioritizing communities to receive IRS based on observed malaria incidence at nearby health centers. Here we show that the use of predicted probabilities of An. funestus to guide IRS implementation saw the largest decrease in malaria incidence at health centers, a 13% reduction (95% confidence interval = 5-21%) compared to concentrating IRS geographically and a 37% reduction (95% confidence interval = 30-44%) compared to targeting IRS based on health facility incidence. These results suggest that vector control programs could produce better outcomes by prioritizing IRS according to malaria-vector risk maps.

Background: Despite rapid upscale of insecticide-treated nets (ITNs) and indoor residual spraying (IRS), malaria remains a major source of morbidity and mortality in Zambia. Uptake and utilization of these and novel interventions are often affected by knowledge, attitudes and practices (KAP) amongst persons living in malaria-endemic areas. The aims of this study were to assess malaria KAP of primary caregivers and explore trends in relation to ITN use, IRS acceptance and mosquito density in two endemic communities in Luangwa and Nyimba districts, Zambia., Methods: A cohort of 75 primary caregivers were assessed using a cross-sectional, forced-choice malaria KAP survey on ITN use, IRS acceptance and initial perception of a novel spatial repellent (SR) product under investigation. Entomological sampling was performed in participant homes using CDC Miniature Light Traps to relate indoor mosquito density with participant responses., Results: Ninety-nine percent of participants cited bites of infected mosquitoes as the route of malaria transmission although other routes were also reported including drinking dirty water (64%) and eating contaminated food (63%). All caregivers agreed that malaria was a life-threatening disease with the majority of caregivers having received malaria information from health centers (86%) and community health workers (51%). Cumulatively, self-reported mosquito net use was 67%. Respondents reportedly liked the SR prototype product but improvements on color, shape and size were suggested. Overall, 398 mosquitoes were captured from light-trap collections, including 49 anophelines and 349 culicines. Insecticide treated nets use was higher in households from which at least one mosquito was captured., Conclusions: The current study identified misconceptions in malaria transmission among primary caregivers indicating remaining knowledge gaps in educational campaigns. Participant responses also indicated a misalignment between a low perception of IRS efficacy and high stated acceptance of IRS, which should be further examined to better understand uptake and sustainability of other vector control strategies. While ITNs were found to be used in study households, misperceptions between presence of mosquitoes and bite protection practices did exist. This study highlights the importance of knowledge attitudes and practice surveys, with integration of entomological sampling, to better guide malaria vector control product development, strategy acceptance and compliance within endemic communities.

Malaria transmission in northern Zambia has increased in the past decade, despite malaria control activities. Evidence-based intervention strategies are needed to effectively reduce malaria transmission. Zambia's National Malaria Control Centre conducted targeted indoor residual spraying (IRS) in Nchelenge District, Luapula Province, from 2014 to 2016 using the organophosphate insecticide pirimiphos-methyl. An evaluation of the IRS campaign was conducted by the Southern Africa International Centers of Excellence for Malaria Research using actively detected malaria cases in bimonthly household surveys carried out from April 2012 to July 2017. Changes in malaria parasite prevalence after IRS were assessed by season using Poisson regression models with robust standard errors, controlling for clustering of participants in households and demographic, geographical, and climatological covariates. In targeted areas, parasite prevalence declined approximately 25% during the rainy season following IRS with pirimiphos-methyl but did not decline during the dry season or in the overall study area. Within targeted areas, parasite prevalence declined in unsprayed households, suggesting both direct and indirect effects of IRS. The moderate decrease in parasite prevalence within sprayed areas indicates that IRS with pirimiphos-methyl is an effective malaria control measure, but a more comprehensive package of interventions is needed to effectively reduce the malaria burden in this setting., (© The Author(s) 2019. Published by Oxford University Press on behalf of the Johns Hopkins Bloomberg School of Public Health. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: While the utility of parasite genotyping for malaria elimination has been extensively documented in low to moderate transmission settings, it has been less well-characterized in holoendemic regions. High malaria burden settings have received renewed attention acknowledging their critical role in malaria elimination. Defining the role for parasite genomics in driving these high burden settings towards elimination will enhance future control programme planning., Methods: Amplicon deep sequencing was used to characterize parasite population genetic diversity at polymorphic Plasmodium falciparum loci, Pfama1 and Pfcsp, at two timepoints in June-July 2016 and January-March 2017 in a high transmission region along the international border between Luapula Province, Zambia and Haut-Katanga Province, the Democratic Republic of the Congo (DRC)., Results: High genetic diversity was observed across both seasons and in both countries. No evidence of population structure was observed between parasite populations on either side of the border, suggesting that this region may be one contiguous transmission zone. Despite a decline in parasite prevalence at the sampling locations in Haut-Katanga Province, no genetic signatures of a population bottleneck were detected, suggesting that larger declines in transmission may be required to reduce parasite genetic diversity. Analysing rare variants may be a suitable alternative approach for detecting epidemiologically important genetic signatures in highly diverse populations; however, the challenge is distinguishing true signals from potential artifacts introduced by small sample sizes., Conclusions: Continuing to explore and document the utility of various parasite genotyping approaches for understanding malaria transmission in holoendemic settings will be valuable to future control and elimination programmes, empowering evidence-based selection of tools and methods to address pertinent questions, thus enabling more efficient resource allocation.

Malaria transmission is dependent on the density and distribution of mosquito vectors, but drivers of vector abundance have not been adequately studied across a range of transmission settings. To inform intervention strategies for high-burden areas, further investigation is needed to identify predictors of vector abundance. Active household (HH) surveillance was conducted in Nchelenge district, Luapula Province, northern Zambia, a high-transmission setting with limited impact of malaria control. Between April 2012 and July 2017, mosquitoes were collected indoors during HH visits using CDC light traps. Demographic, environmental, and climatological correlates of vector abundance were identified using log-binomial regression models with robust standard errors. The primary malaria vectors in this setting were Anopheles funestus sensu stricto (s.s.) and Anopheles gambiae s.s. Anopheles funestus predominated in both seasons, with a peak in the dry season. Anopheles gambiae peaked at lower numbers in the rainy season. Environmental, climatic, and demographic factors were correlated with HH vector abundance. Higher vector counts were found in rural areas with low population density and among HHs close to roads and small streams. Vector counts were lower with increasing elevation and slope. Anopheles funestus was negatively associated with rainfall at lags of 2-6 weeks, and An. gambiae was positively associated with rainfall at lags of 3-10 weeks. Both vectors had varying relationships with temperature. These results suggest that malaria vector control in Nchelenge district should occur throughout the year, with an increased focus on dry-season transmission and rural areas.

Background: Southern Province, Zambia has experienced a dramatic decline in Plasmodium falciparum malaria transmission in the past decade and is targeted for elimination. Zambia's National Malaria Elimination Program recommends reactive case detection (RCD) within 140 m of index households to enhance surveillance and eliminate remaining transmission foci., Methods: To evaluate whether RCD captures local transmission, we genotyped 26 microsatellites from 106 samples collected from index (n = 27) and secondary (n = 79) cases detected through RCD in the Macha Hospital catchment area between January 2015 and April 2016., Results: Participants from the same RCD event harbored more genetically related parasites than those from different RCD events, suggesting that RCD captures, at least in part, infections related through local transmission. Related parasites clustered in space and time, up to at least 250 m from index households. Spatial analysis identified a putative focal transmission hotspot., Conclusions: The current RCD strategy detects focal transmission events, although programmatic guidelines to screen within 140 m of index households may fail to capture all secondary cases. This study highlights the utility of parasite genetic data in assessing programmatic interventions, and similar approaches may be useful to malaria elimination programs seeking to tailor intervention strategies to the underlying transmission epidemiology., (© The Author(s) 2018. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

Antibodies to Plasmodium falciparum are specific biomarkers that can be used to monitor parasite exposure over broader time frames than microscopy, rapid diagnostic tests, or molecular assays. Consequently, seroprevalence surveys can assist with monitoring the impact of malaria control interventions, particularly in the final stages of elimination, when parasite incidence is low. The protein array format to measure antibodies to diverse P. falciparum antigens requires only small sample volumes and is high throughput, permitting the monitoring of malaria transmission on large spatial and temporal scales. We expanded the use of a protein microarray to assess malaria transmission in settings beyond those with a low malaria incidence. Antibody responses in children and adults were profiled, using a P. falciparum protein microarray, through community-based surveys in three areas in Zambia and Zimbabwe at different stages of malaria control and elimination. These three epidemiological settings had distinct serological profiles reflective of their malaria transmission histories. While there was little correlation between transmission intensity and antibody signals (magnitude or breadth) in adults, there was a clear correlation in children younger than 5 years of age. Antibodies in adults appeared to be durable even in the absence of significant recent transmission, whereas antibodies in children provided a more accurate picture of recent levels of transmission intensity. Seroprevalence studies in children could provide a valuable marker of progress toward malaria elimination. IMPORTANCE As malaria approaches elimination in many areas of the world, monitoring the effect of control measures becomes more important but challenging. Low-level infections may go undetected by conventional tests that depend on parasitemia, particularly in immune individuals, who typically show no symptoms of malaria. In contrast, antibodies persist after parasitemia and may provide a more accurate picture of recent exposure. Only a few parasite antigens-mainly vaccine candidates-have been evaluated in seroepidemiological studies. We examined antibody responses to 500 different malaria proteins in blood samples collected through community-based surveillance from areas with low, medium, and high malaria transmission intensities. The breadth of the antibody responses in adults was broad in all three settings and was a poor correlate of recent exposure. In contrast, children represented a better sentinel population for monitoring recent malaria transmission. These data will help inform the use of multiplex serology for malaria surveillance., (Copyright © 2019 Kobayashi et al.)

A large proportion of ongoing malaria parasite transmission is attributed to low-density subclinical infections not readily detected by available rapid diagnostic tests (RDTs) or microscopy. Plasmodium falciparum gametocyte carriage is subclinical, but gametocytemic individuals comprise the parasite reservoir that leads to infection of mosquitoes and local transmission. Effective detection and quantification of these carriers can help advance malaria elimination strategies. However, no point-of-need (PON) RDTs for gametocyte detection exist, much less one that can perform noninvasive sampling of saliva outside a clinical setting. Here, we report on the discovery of 35 parasite markers from which we selected a single candidate for use in a PON RDT. We performed a cross-sectional, multi-omics study of saliva from 364 children with subclinical infection in Cameroon and Zambia and produced a prototype saliva-based PON lateral flow immunoassay test for P. falciparum gametocyte carriers. The test is capable of identifying submicroscopic carriage in both clinical and nonclinical settings and is compatible with archived saliva samples., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

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.

To improve malaria surveillance and achieve elimination, the Zambian National Malaria Elimination Program implemented a reactive test-and-treat program in Southern Province in 2013 in which individuals with rapid diagnostic test (RDT)-confirmed malaria are followed-up at their home within 1 week of diagnosis. Individuals present at the index case household and those residing within 140 m of the index case are tested with an RDT and treated with artemether-lumefantrine if positive. This study evaluated the efficiency of this reactive test-and-treat strategy by characterizing infected individuals missed by the RDT and the current screening radius. The radius was expanded to 250 m, and a quantitative polymerase chain reaction (qPCR) test was performed on dried blood spot specimens. From January 2015 through March 2016, 145 index cases were identified at health centers and health posts. A total of 3,333 individuals residing in 525 households were screened. Excluding index cases, the parasite prevalence was 1.1% by RDT (33 positives of 3,016 participants) and 2.4% by qPCR (73 positives of 3,016 participants). Of the qPCR-positive cases, 62% of 73 individuals tested negative by RDT. Approximately half of the infected individuals resided within the index case household (58% of RDT-positive individuals and 48% of qPCR-positive individuals). The low sensitivity of the RDT and the high proportion of secondary cases within the index case household decreased the efficiency of this reactive test-and-treat strategy. Reactive focal drug administration in index case households would be a more efficient approach to treating infected individuals associated with a symptomatic case.

The RTS,S/AS01 malaria vaccine encompasses the central repeats and C-terminal of Plasmodium falciparum circumsporozoite protein (PfCSP). Although no Phase II clinical trial studies observed evidence of strain-specific immunity, recent studies show a decrease in vaccine efficacy against non-vaccine strain parasites. In light of goals to reduce malaria morbidity, anticipating the effectiveness of RTS,S/AS01 is critical to planning widespread vaccine introduction. We deep sequenced C-terminal Pfcsp from 77 individuals living along the international border in Luapula Province, Zambia and Haut-Katanga Province, the Democratic Republic of the Congo (DRC) and compared translated amino acid haplotypes to the 3D7 vaccine strain. Only 5.2% of the 193 PfCSP sequences from the Zambia-DRC border region matched 3D7 at all 84 amino acids. To further contextualize the genetic diversity sampled in this study with global PfCSP diversity, we analyzed an additional 3,809 Pfcsp sequences from the Pf3k database and constructed a haplotype network representing 15 countries from Africa and Asia. The diversity observed in our samples was similar to the diversity observed in the global haplotype network. These observations underscore the need for additional research assessing genetic diversity in P. falciparum and the impact of PfCSP diversity on RTS,S/AS01 efficacy.

Understanding factors influencing sustained use of long-lasting insecticide-treated nets (LLIN) in areas of declining malaria transmission is critical to sustaining control and may facilitate elimination. From 2008 to 2013, 655 households in Choma District, Zambia, were randomly selected and residents were administered a questionnaire and malaria rapid diagnostic test. Mosquitoes were collected concurrently by light trap. In a multilevel model, children and adolescents of 5-17 years of age were 55% less likely to sleep under LLIN than adults (odds ratio [OR] = 0.45; 95% confidence interval [CI] = 0.35, 0.58). LLIN use was 80% higher during the rainy season (OR = 1.8; CI = 1.5, 2.2) and residents of households with three or more nets were over twice as likely to use a LLIN (OR = 2.1; CI = 1.4, 3.1). For every increase in 0.5 km from the nearest health center, the odds of LLIN use decreased 9% (OR = 0.9; CI = 0.88, 0.98). In a second multilevel model, the odds of LLIN use were more than twice high if more than five mosquitoes (anopheline and culicine) were captured in the house compared with households with no mosquitoes captured (OR = 2.1; CI = 1.1, 3.9). LLIN use can be sustained in low-transmission settings with continued education and distributions, and may be partially driven by the presence of nuisance mosquitoes., (© The American Society of Tropical Medicine and Hygiene.)

Background: Defining the anopheline mosquito vectors and their foraging behaviour in malaria endemic areas is crucial for disease control and surveillance. The standard protocol for molecular identification of host blood meals in mosquitoes is to morphologically identify fed mosquitoes and then perform polymerase chain reaction (PCR), precipitin tests, or ELISA assays. The purpose of this study was to determine the extent to which the feeding rate and human blood indices (HBIs) of malaria vectors were underestimated when molecular confirmation by PCR was performed on both visually fed and unfed mosquitoes., Methods: In association with the Southern Africa International Centers of Excellence in Malaria Research (ICEMR), mosquito collections were performed at three sites: Choma district in southern Zambia, Nchelenge district in northern Zambia, and Mutasa district in eastern Zimbabwe. All anophelines were classified visually as fed or unfed, and tested for blood meal species using PCR methods. The HBIs of visually fed mosquitoes were compared to the HBIs of overall PCR confirmed fed mosquitoes by Pearson's Chi-Square Test of Independence., Results: The mosquito collections consisted of Anopheles arabiensis from Choma, Anopheles funestus s.s., Anopheles gambiae s.s. and Anopheles leesoni from Nchelenge, and An. funestus s.s. and An. leesoni from Mutasa. The malaria vectors at all three sites had large human blood indices (HBI) suggesting high anthropophily. When only visually fed mosquitoes tested by PCR for blood meal species were compared to testing those classified as both visually fed and unfed mosquitoes, it was found that the proportion blooded was underestimated by up to 18.7%. For most Anopheles species at each site, there was a statistically significant relationship (P < 0.05) between the HBIs of visually fed mosquitoes and that of the overall PCR confirmed fed mosquitoes., Conclusion: The impact on HBI of analysing both visually fed and unfed mosquitoes varied from site to site. This discrepancy may be due to partial blood feeding behaviour by mosquitoes, digestion of blood meals, sample condition, and/or expertise of entomology field staff. It is important to perform molecular testing on all mosquitoes to accurately characterize vector feeding behaviour and develop interventions in malaria endemic areas.

In 2007, the first free mass distribution of insecticide-treated bed nets (ITNs) occurred in southern Zambia. To determine the effect of ITNs on heterogeneity in biting rates, human DNA from Anopheles arabiensis blood meals was genotyped to determine the number of hosts that had contributed to the blood meals. The multiple feeding rate decreased from 18.9% pre-ITN to 9.1% post-ITN, suggesting that mosquito biting had focused onto a smaller fraction of the population. Pre-ITN, 20% of persons in a household provided 40% of blood meals, which increased to 59% post-ITN. To measure heterogeneity over a larger scale, mosquitoes were collected in 90 households in two village areas. Of these households, 25% contributed 78.1% of An. arabiensis, and households with high frequencies of An. arabiensis were significantly spatially clustered. The results indicate that substantial heterogeneity in malaria risk exists at local and household levels, and household-level heterogeneity may be influenced by interventions, such as ITNs.

Anopheles coustani s.l. and Anopheles squamosus are sub-Saharan mosquito species that have been implicated in malaria transmission. Although generally believed to be of negligible importance due to their overwhelmingly zoophilic behavior, An. coustani s.l. and An. squamosus made up a large proportion of the anophelines collected by human landing catches during the 2007-2008 and 2008-2009 rainy seasons in Macha, Zambia. Further, polymerase chain reaction-based blood meal identification showed that the majority of blood meals from these mosquito species caught in human-baited Centers for Disease Control light traps were from human hosts. Although no An. coustani s.l. or An. squamosus were found to be positive for Plasmodium, the demonstrated anthropophilic tendencies of these mosquitoes in southern Zambia suggest their potential as secondary malaria vectors.

Human landing catches (HLCs) are currently the preferred method to determine vector human biting rates (HBRs), which are key determinants of entomologic inoculation rates and important measures for assessing the impact of vector control efforts. Although HLCs are the most direct means of establishing HBRs, they are labor-intensive, and their use is facing increasing ethical concerns. The relationship between Centers for Disease Control (CDC) light traps and HLC collections was evaluated in Macha, Zambia during the 2007-2008 and 2008-2009 rainy seasons. A CDC light trap captured on average 1.91 (95% confidence interval = 1.16-2.28) times as many An. arabiensis per night as an indoor HLC. Additionally, nets treated with deltamethrin did not affect the numbers of An. arabiensis collected. Our results suggest that in regions where use of vector control interventions is high and vector densities are low, CDC light traps can be used to monitor An. arabiensis HBRs.

Anopheles arabiensis mosquitoes are the primary vector responsible for Plasmodium falciparum transmission in Macha, Zambia. Because insecticide-treated bed nets (ITNs) have the potential to alter host feeding behavior, the extent of the zoophilic and exophagic tendencies of the vector was evaluated during the two rainy seasons after ITN introduction. Centers for Disease Control light traps, paired indoor/outdoor human landing catches, and outdoor cattle-baited collections were used to assess potential changes in host preference. Results support the hypothesis that An. arabiensis mosquitoes in Macha remain highly anthropophilic despite high ITN use. Anopheles arabiensis mosquitoes in Macha appear to be relatively exophagic and have been caught biting outdoors immediately after sunset and before sunrise, potentially circumventing some of the protective effects of ITNs.

Anopheles arabiensis is a major vector of Plasmodium falciparum in southern Zambia. This study aimed to determine the rate of multiple human blood meals taken by An. arabiensis to more accurately estimate entomologic inoculation rates (EIRs). Mosquitoes were collected in four village areas over two seasons. DNA from human blood meals was extracted and amplified at four microsatellite loci. Using the three-allele method, which counts > or = 3 alleles at any microsatellite locus as a multiple blood meal, we determined that the overall frequency of multiple blood meals was 18.9%, which was higher than rates reported for An. gambiae in Kenya and An. funestus in Tanzania. Computer simulations showed that the three-allele method underestimates the true multiple blood meal proportion by 3-5%. Although P. falciparum infection status was not shown to influence the frequency of multiple blood feeding, the high multiple feeding rate found in this study increased predicted malaria risk by increasing EIR.

An increase in Anopheles arabiensis showing endophilic behavior was observed in Macha, Zambia during March 2007. To determine whether this shift in resting behavior was accompanied by a change in feeding preference, an attempt was made to calculate the human blood index. However, only 46.2% of blood meals were successfully identified with existing polymerase chain reaction (PCR) diagnostics. This failure was hypothesized to be caused by the limitations of existing methods that are not capable of identifying host source from anophelines resting for extended time periods. Using an assay we developed that allows for the identification of mammalian host DNA out to 60 hours post-feeding, we were able to successfully determine the host source of 94.3% of recovered blood meals. The data show that, although An. arabiensis in Macha experienced a period of higher endophily, the degree of anthropophily and the sporozoite rate in the population remained comparable to the previous malaria season.

No studies have addressed the spatial complexity of Anopheles arabiensis populations in Zambia or the effects of drought on the genetic structure of this species. We genotyped approximately 420 An. arabiensis at 12 microsatellite loci representing 18 collections from the Southern Province of Zambia. Collections spanned three transmission seasons and covered a wet year-drought year-wet year cycle. Anopheles arabiensis within the 2,000 km(2) of the Macha study region were panmictic, with high gene flow between Macha and Namwala, Zambia, which are 80 km apart. There was little evidence for genetic structuring among years, with no significant shifts in allele frequency distributions or observed heterozygosity, and no evidence for a genetic bottleneck despite a drastic reduction in mosquito numbers during the drought year. Anopheles arabiensis in southern Zambia has a large deme size, and the regional genetic structure of this species was little affected by an extended drought period.

Genetic mutations controlling eye color, fat body color, structural abnormalities, and insecticide resistance are common in mosquitoes. We have identified a novel color variant of Culex pipiens quinquefasciatus characterized by a heavily pigmented integument in adult specimens circulating in field populations of this species in southern Zambia. Mosquitoes were collected monthly by pyrethrum spray catch between November and May 2004-05 and 2005-06, with between 25% and 80% of the total Cx. p. quinquefasciatus collections comprising this pigmented variety. The identity of pigmented specimens was morphologically confirmed as Cx. p. quinquefasciatus by examination of orientation of the dorsal and ventral arms of the male genitalia of F1 male progeny reared in the laboratory, and molecularly verified by diagnostic polymerase chain reaction and nicotinamide adenine dinucleotide phosphate dehydrogenase subunit 4 nucleotide sequence homology. Preliminary laboratory rearings indicated that the pigmentation was heritable and not influenced by larval habitat and environmental conditions. Further investigation into the mechanism of the mutation, inheritance patterns, and potential linkage to additional markers is pending.

Transmission of Plasmodium falciparum is hyperendemic in southern Zambia. However, no data on the entomologic aspects of malaria transmission have been published from Zambia in more than 25 years. We evaluated seasonal malaria transmission by Anopheles arabiensis and An. funestus s.s. and characterized the blood feeding behavior of An. arabiensis in two village areas. Transmission during the 2004-2005 rainy season was nearly zero because of widespread drought. During 2005-2006, the estimated entomologic inoculation rate values were 1.6 and 18.3 infective bites per person per transmission season in each of the two village areas, respectively. Finally, with a human blood index of 0.923, An. arabiensis was substantially more anthropophilic in our study area than comparable samples of indoor-resting An. arabiensis throughout Africa and was the primary vector responsible for transmission of P. falciparum.

Anopheles longipalpis (Theobald) (Diptera: Culicidae) is a predominantly zoophilic mosquito that has not been implicated in malaria transmission. However, this species was collected indoors with An. funestus s.l. in southern Zambia, where transmission of Plasmodium falciparum is hyperendemic, and we initially misidentified it morphologically and molecularly as An. funestus s.l. The indoor resting density and blood-feeding behaviour of An. longipalpis were investigated during the 2004-05 and 2005-06 transmission seasons in Mufwafwi village in southern Zambia. Numbers of endophilic An. longipalpis increased towards the end of the rainy season. Although specimens were collected during human landing catches, the feeding behaviour of An. longipalpis was significantly biased towards cattle (88.7%), with other bloodmeals originating from dogs, goats and chickens. None of the 177 specimens of An. longipalpis were infected with P. falciparum. These data are consistent with existing reports that An. longipalpis is not involved in malaria transmission. However, more extensive sampling is necessary. Importantly, the correct identification of An. longipalpis is crucial for malaria control programmes in areas where An. funestus s.l and An. longipalpis exist sympatrically so that scarce resources are not wasted on the control of a non-vector.