Temporal genomic analysis of Plasmodium falciparum reveals increased prevalence of mutations associated with delayed clearance following treatment with artemisinin-lumefantrine in Choma District, Southern Province, Zambia.

The emergence of antimalarial drug resistance is an impediment to malaria control and elimination in Africa. Analysis of temporal trends in molecular markers of resistance is critical to inform policy makers and guide malaria treatment guidelines. In a low and seasonal transmission region of southern Zambia, we successfully genotyped 85.5% (389/455) of Plasmodium falciparum samples collected between 2013-2018 from 8 spatially clustered health centres using molecular inversion probes (MIPs) targeting key drug resistance genes. Aside from one sample carrying K13 R622 I , none of the isolates carried other World Health Organization-validated or candidate artemisinin partial resistance (ART-R) mutations in K13. However, 13% (CI, 9.6-17.2) of isolates had the AP2MU S160 N mutation, which has been associated with delayed clearance following artemisinin combination therapy in Africa. This mutation increased in prevalence between 2015-2018 and bears a genomic signature of selection. During this time period, there was an increase in the MDR1 N F D haplotype that is associated with reduced susceptibility to lumefantrine. Sulfadoxine-pyrimethamine polymorphisms were near fixation. While validated ART-R mutations are rare, a mutation associated with slow parasite clearance in Africa appears to be under selection in southern Zambia.

A metagenomic analysis of the phase 2 Anopheles gambiae 1000 genomes dataset reveals a wide diversity of cobionts associated with field collected mosquitoes.

The Anopheles gambiae 1000 Genomes (Ag1000G) Consortium previously utilized deep sequencing methods to catalogue genetic diversity across African An. gambiae populations. We analyzed the complete datasets of 1142 individually sequenced mosquitoes through Microsoft Premonition's Bayesian mixture model based (BMM) metagenomics pipeline. All specimens were confirmed as either An. gambiae sensu stricto (s.s.) or An. coluzzii with a high degree of confidence ( > 98% identity to reference). Homo sapiens DNA was identified in all specimens indicating contamination may have occurred either at the time of specimen collection, preparation and/or sequencing. We found evidence of vertebrate hosts in 162 specimens. 59 specimens contained validated Plasmodium falciparum reads. Human hepatitis B and primate erythroparvovirus-1 viral sequences were identified in fifteen and three mosquito specimens, respectively. 478 of the 1,142 specimens were found to contain bacterial reads and bacteriophage-related contigs were detected in 27 specimens. This analysis demonstrates the capacity of metagenomic approaches to elucidate important vector-host-pathogen interactions of epidemiological significance.

Genomics reveals heterogeneous Plasmodium falciparum transmission and selection signals in Zambia.

BACKGROUND: Genomic surveillance is crucial for monitoring malaria transmission and understanding parasite adaptation to interventions. Zambia lacks prior nationwide efforts in malaria genomic surveillance among African countries. METHODS: We conducted genomic surveillance of Plasmodium falciparum parasites from the 2018 Malaria Indicator Survey in Zambia, a nationally representative household survey of children under five years of age. We whole-genome sequenced and analyzed 241 P. falciparum genomes from regions with varying levels of malaria transmission across Zambia and estimated genetic metrics that are informative about transmission intensity, genetic relatedness between parasites, and selection. RESULTS: We provide genomic evidence of widespread within-host polygenomic infections, regardless of epidemiological characteristics, underscoring the extensive and ongoing endemic malaria transmission in Zambia. Our analysis reveals country-level clustering of parasites from Zambia and neighboring regions, with distinct separation in West Africa. Within Zambia, identity by descent (IBD) relatedness analysis uncovers local spatial clustering and rare cases of long-distance sharing of closely related parasite pairs. Genomic regions with large shared IBD segments and strong positive selection signatures implicate genes involved in sulfadoxine-pyrimethamine and artemisinin combination therapies drug resistance, but no signature related to chloroquine resistance. Furthermore, differences in selection signatures, including drug resistance loci, are observed between eastern and western Zambian parasite populations, suggesting variable transmission intensity and ongoing drug pressure. CONCLUSIONS: Our findings enhance our understanding of nationwide P. falciparum transmission in Zambia, establishing a baseline for analyzing parasite genetic metrics as they vary over time and space. These insights highlight the urgency of strengthening malaria control programs and surveillance of antimalarial drug resistance.

Malaria is caused by a parasite that is spread to humans via mosquito bites. It is a leading cause of death in children under five years old in sub-Saharan Africa. Analysis of the malaria parasite's complete set of DNA (its genome) can help us to understand transmission of the disease and how this changes in response to different strategies to control the disease. We analyzed the genomes of malaria parasites from children across Zambia. Our study revealed that 77% of children harbored multiple parasite strains, which suggests that local transmission (transmission between people within the same local area) is high. Genetic evidence for long-distance transmission was rarer. Furthermore, our findings suggest parasites are evolving in response to antimalarial drugs. Our study enhances our understanding of malaria dynamics in Zambia and may help to inform strategies for improved surveillance and control.

Genomics reveals heterogeneous Plasmodium falciparum transmission and population differentiation in Zambia and bordering countries.

Genomic surveillance plays a critical role in monitoring malaria transmission and understanding how the parasite adapts in response to interventions. We conducted genomic surveillance of malaria by sequencing 241 Plasmodium falciparum genomes from regions with varying levels of malaria transmission across Zambia. We found genomic evidence of high levels of within-host polygenomic infections, regardless of epidemiological characteristics, underscoring the extensive and ongoing endemic malaria transmission in the country. We identified country-level clustering of parasites from Zambia and neighboring countries, and distinct clustering of parasites from West Africa. Within Zambia, our identity by descent (IBD) relatedness analysis uncovered spatial clustering of closely related parasite pairs at the local level and rare cases of long-distance sharing. Genomic regions with large shared IBD segments and strong positive selection signatures identified genes involved in sulfadoxine-pyrimethamine and artemisinin combination therapies drug resistance, but no signature related to chloroquine resistance. Together, our findings enhance our understanding of P. falciparum transmission nationwide in Zambia and highlight the urgency of strengthening malaria control programs and surveillance of antimalarial drug resistance.

The In Vitro Antimicrobial Susceptibility of Borrelia burgdorferi sensu lato: Shedding Light on the Known Unknowns.

Human Lyme borreliosis (LB) represents a multisystem disorder that can progress in stages. The causative agents are transmitted by hard ticks of the Ixodes ricinus complex that have been infected with the spirochete Borrelia burgdorferi sensu lato. Today, LB is considered the most important human tick-borne illness in the Northern Hemisphere. The causative agent was identified and successfully isolated in 1982 and, shortly thereafter, antibiotic treatment was found to be safe and efficacious. Since then, various in vitro studies have been conducted in order to improve our knowledge of the activity of antimicrobial agents against B. burgdorferi s. l. The full spectrum of in vitro antibiotic susceptibility has still not been defined for some of the more recently developed compounds. Moreover, our current understanding of the in vitro interactions between B. burgdorferi s. l. and antimicrobial agents, and their possible mechanisms of resistance remains very limited and is largely based on in vitro susceptibility experiments on only a few isolates of Borrelia. Even less is known about the possible mechanisms of the in vitro persistence of spirochetes exposed to antimicrobial agents in the presence of human and animal cell lines. Only a relatively small number of laboratory studies and cell culture experiments have been conducted. This review summarizes what is and what is not known about the in vitro susceptibility of B. burgdorferi s. l. It aims to shed light on the known unknowns that continue to fuel current debates on possible treatment resistance and mechanisms of persistence of Lyme disease spirochetes in the presence of antimicrobial agents.

Spatial-temporal vector abundance and malaria transmission dynamics in Nchelenge and Lake Mweru islands, a region with a high burden of malaria in northern Zambia.

BACKGROUND: Over a decade of vector control by indoor residual spraying (IRS) and long-lasting insecticidal nets (LLINs) distribution on the mainland, and only LLINs on islands had a minimal impact on disease burden in Nchelenge district, northern Zambia. Anopheles funestus and Anopheles gambiae are vectors known only from the mainland. Understanding vector bionomics in the district is necessary for planning and targeting effective vector control. This study aimed to provide information on abundance, seasonality, and Plasmodium falciparum sporozoite infectivity of malaria vectors in Nchelenge, including islands. METHODS: Mosquitoes were collected in 192 CDC indoor light traps set in 56 households between January 2015 and January 2016. Morphological and molecular species identifications and P. falciparum circumsporoites by ELISA were performed. Mosquito counts and relative abundances from the islands and mainland were compared, and household factors associated with vector counts were determined. RESULTS: A total of 5888 anophelines were collected during the study. Of these, 5,704 were female Anopheles funestus sensu lato (s.l.) and 248 female An. gambiae s.l. The highest proportion of An. funestus (n = 4090) was from Chisenga Island and An. gambiae (n = 174) was from Kilwa Island. The highest estimated counts per trap for An. funestus s.l. were from Chisenga island, (89.9, p < 0.001) and from the dry season (78.6, p < 001). For An. gambiae the highest counts per trap were from Kilwa island (3.1, p < 0.001) and the rainy season (2.5, p = 0.007). The highest estimated annual entomological inoculation rate was from Chisenga Island with 91.62 ib/p/y followed by Kilwa Island with 29.77 ib/p/yr, and then Mainland with 19.97 ib/p/yr. CONCLUSIONS: There was varied species abundance and malaria transmission risk across sites and seasons. The risk of malaria transmission was perennial and higher on the islands. The minimal impact of vector control efforts on the mainland was evident, but limited overall. Vector control intervention coverage with effective tools needs to be extended to the islands to effectively control malaria transmission in Nchelenge district.

The first genome sequence of Anopheles squamous from Macha, Zambia.

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.

Novel bioinformatic methods and machine learning approaches reveal candidate biomarkers of the intensity and timing of past exposure to Plasmodium falciparum.

Accurately quantifying the burden of malaria over time is an important goal of malaria surveillance efforts and can enable effective targeting and evaluation of interventions. Malaria surveillance methods capture active or recent infections which poses several challenges to achieving malaria surveillance goals. In high transmission settings, asymptomatic infections are common and therefore accurate measurement of malaria burden demands active surveillance; in low transmission regions where infections are rare accurate surveillance requires sampling large subsets of the population; and in any context monitoring malaria burden over time necessitates serial sampling. Antibody responses to Plasmodium falciparum parasites persist after infection and therefore measuring antibodies has the potential to overcome several of the current obstacles to accurate malaria surveillance. Identifying which antibody responses are markers of the timing and intensity of past exposure to P. falciparum remains challenging, particularly among adults who tend to be re-exposed multiple times over the course of their lifetime and therefore have similarly high antibody responses to many Plasmodium antigens. A previous analysis of 479 serum samples from individuals in three regions in southern Africa with different historical levels of P. falciparum malaria transmission (high, intermediate, and low) revealed regional differences in antibody responses to P. falciparum antigens among children under 5 years of age. Using a novel bioinformatic pipeline optimized for protein microarrays that minimizes between-sample technical variation, we used antibody responses to Plasmodium antigens as predictors in random forest models to classify samples from adults into these three regions of differing historical malaria transmission with high accuracy (AUC = 0.99). Many of the most important antigens for classification in these models do not overlap with previously published results and are therefore novel candidate markers for the timing and intensity of past exposure to P. falciparum. Measuring antibody responses to these antigens could lead to improved malaria surveillance.

Temporal and spatial analysis of Plasmodium falciparum genomics reveals patterns of parasite connectivity in a low-transmission district in Southern Province, Zambia.

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.

The Impact of Household and Community Indoor Residual Spray Coverage with Fludora Fusion in a High Malaria Transmission Setting in Northern Zambia.

Zambia's National Malaria Elimination Program transitioned to Fludora Fusion in 2019 for annual indoor residual spraying (IRS) in Nchelenge District, an area with holoendemic malaria transmission. Previously, IRS was associated with reductions in parasite prevalence during the rainy season only, presumably because of insufficient residual insecticide longevity. This study assessed the impact of transitioning from Actellic 300CS to long-acting Fludora Fusion using active surveillance data from 2014 through 2021. A difference-in-differences analysis estimated changes in rainy season parasite prevalence associated with living in a sprayed house, comparing insecticides. The change in the 2020 to 2021 dry season parasite prevalence associated with living in a house sprayed with Fludora Fusion was also estimated. Indoor residual spraying with Fludora Fusion was not associated with decreased rainy season parasite prevalence compared with IRS with Actellic 300CS (ratio of prevalence ratios [PRs], 1.09; 95% CI, 0.89-1.33). Moreover, living in a house sprayed with either insecticide was not associated with decreased malaria risk (Actellic 300CS: PR, 0.97; 95% CI, 0.86-1.10; Fludora Fusion: rainy season PR, 1.06; 95% CI, 0.89-1.25; dry season PR, 1.21; 95% CI, 0.99-1.48). In contrast, each 10% increase in community IRS coverage was associated with a 4% to 5% reduction in parasite prevalence (rainy season: PR, 0.95; 95% CI, 0.92-0.97; dry season: PR, 0.96; 95% CI, 0.94-0.99), suggesting a community-level protective effect, and corroborating the importance of high-intervention coverage.

A Bayesian hierarchical model for signal extraction from protein microarrays.

Protein microarrays are a promising technology that measure protein levels in serum or plasma samples. Due to their high technical variability and high variation in protein levels across serum samples in any population, directly answering biological questions of interest using protein microarray measurements is challenging. Analyzing preprocessed data and within-sample ranks of protein levels can mitigate the impact of between-sample variation. As for any analysis, ranks are sensitive to preprocessing, but loss function based ranks that accommodate major structural relations and components of uncertainty are very effective. Bayesian modeling with full posterior distributions for quantities of interest produce the most effective ranks. Such Bayesian models have been developed for other assays, for example, DNA microarrays, but modeling assumptions for these assays are not appropriate for protein microarrays. Consequently, we develop and evaluate a Bayesian model to extract the full posterior distribution of normalized protein levels and associated ranks for protein microarrays, and show that it fits well to data from two studies that use protein microarrays produced by different manufacturing processes. We validate the model via simulation and demonstrate the downstream impact of using estimates from this model to obtain optimal ranks.

Scientific Findings of the Southern and Central Africa International Center of Excellence for Malaria Research: Ten Years of Malaria Control Impact Assessments in Hypo-, Meso-, and Holoendemic Transmission Zones in Zambia and Zimbabwe.

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.

Policy Implications of the Southern and Central Africa International Center of Excellence for Malaria Research: Ten Years of Malaria Control Impact Assessments in Hypo-, Meso-, and Holoendemic Transmission Zones in Zambia and Zimbabwe.

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.

Expanded geographic distribution and host preference of Anopheles gibbinsi (Anopheles species 6) in northern Zambia.

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.

Understudied Anophelines Contribute to Malaria Transmission in a Low-Transmission Setting in the Choma District, Southern Province, Zambia.

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.

Current knowledge of vector-borne zoonotic pathogens in Zambia: A clarion call to scaling-up "One Health" research in the wake of emerging and re-emerging infectious diseases.

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.

A pre-processing pipeline to quantify, visualize, and reduce technical variation in protein microarray studies.

Technical variation, or variation from non-biological sources, is present in most laboratory assays. Correcting for this variation enables analysts to extract a biological signal that informs questions of interest. However, each assay has different sources and levels of technical variation, and the choice of correction methods can impact downstream analyses. Compared to similar assays such as DNA microarrays, relatively few methods have been developed and evaluated for protein microarrays, a versatile tool for measuring levels of various proteins in serum samples. Here, we propose a pre-processing pipeline to correct for some common sources of technical variation in protein microarrays. The pipeline builds upon an existing normalization method by using controls to reduce technical variation. We evaluate our method using data from two protein microarray studies and by simulation. We demonstrate that pre-processing choices impact the fluorescent-intensity based ranks of proteins, which in turn, impact downstream analysis.

Phylogenetic Complexity of Morphologically Identified Anopheles squamosus in Southern Zambia.

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.

Multiple Novel Clades of Anopheline Mosquitoes Caught Outdoors in Northern 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.

The Impact of Three Years of Targeted Indoor Residual Spraying with Pirimiphos-Methyl on Household Vector Abundance in a High Malaria Transmission Area of Northern Zambia.

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.