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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.

BACKGROUND: Southern Province, Zambia has experienced a dramatic decline in Plasmodium falciparum malaria transmission in the past decade and is targeted for elimination. Zambias 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.

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. [ABSTRACT FROM AUTHOR]

Abstract Background Critical to sustaining progress in malaria control is comprehensive surveillance to identify outbreaks and prevent resurgence. Serologic responses to Plasmodium falciparum antigens can serve as a marker of recent transmission and serosurveillance may be feasible on a large scale. Methods Satellite images were used to construct a sampling frame for the random selection of households enrolled in prospective longitudinal and cross-sectional surveys in two study areas in Southern Province, Zambia, one in 2007 and the other in 2008 and 2009. Blood was collected and stored as dried spots from participating household members. A malaria rapid diagnostic test (RDT) was used to diagnose malaria. An enzyme immunoassay (EIA) was used to detect IgG antibodies to asexual stage P. falciparum whole parasite lysate using serum eluted from dried blood spots. The expected mean annual increase in optical density (OD) value for individuals with a documented prior history of recent malaria was determined using mixed models. SatScan was used to determine the spatial clustering of households with individuals with serological evidence of recent malaria, and these households were plotted on a malaria risk map. Results RDT positivity differed markedly between the study areas and years: 28% of participants for whom serologic data were available were RDT positive in the 2007 study area, compared to 8.1% and 1.4% in the 2008 and 2009 study area, respectively. Baseline antibody levels were measured in 234 participants between April and July 2007, 435 participants between February and December 2008, and 855 participants between January and December 2009. As expected, the proportion of seropositive individuals increased with age in each year. In a subset of participants followed longitudinally, RDT positivity at the prior visit was positively correlated with an increase in EIA OD values after adjusting for age in 2007 (0.261, p = 0.003) and in 2008 (0.116, p = 0.03). RDT positivity at the concurrent visit also was associated with an increase in EIA OD value in 2007 (mean increase 0.177, p = 0.002) but not in 2008 (−0.063, p =0.50). Households comprised of individuals with serologic evidence of recent malaria overlapped areas of high malaria risk for serologic data from 2009, when parasite prevalence was lowest. Conclusions Serological surveys to whole asexual P. falciparum antigens using blood collected as dried blood spots can be used to detect temporal and spatial patterns of malaria transmission in a region of declining malaria burden, and have the potential to identify focal areas of recent transmission.

Abstract Background Malaria elimination will require that both symptomatic- and asymptomatic-infected persons be identified and treated. However, well-characterized, individual-level risk factors for malaria may not be valid in regions with declining malaria transmission. Changes in individual-level correlates of malaria infection were evaluated over three years in a region of declining malaria transmission in southern Zambia. Methods Malaria surveys were conducted in two study areas within the catchment area of Macha Hospital, Zambia in 2007 and 2008/2009. A random sample of households was identified from a digitized satellite image of the study areas. Cross-sectional surveys were conducted approximately five times throughout the year in each of the two study areas. During study visits, adults and caretakers of children were administered questionnaires and a blood sample was obtained for a rapid diagnostic test (RDT) for malaria. Results In the 2007 study area, 330 individuals were surveyed. 40.9% of participants lived in a household with at least one insecticide-treated bed net (ITN); however, only 45.2% reported sleeping under the ITN. 23.9% of participants were RDT positive. Correlates of RDT positivity included younger age, the presence of symptoms, testing during the rainy season, using an open water source, and not sleeping under an ITN. In the 2008 study area, 435 individuals were surveyed. 77.0% of participants lived in a household with at least one ITN; however, only 56.4% reported sleeping under the ITN. 8.1% of participants were RDT positive. RDT positivity was negatively correlated with the presence of symptoms within the last two weeks but positively correlated with documented fever. In 2009, 716 individuals were surveyed in the same area as 2008. 63.7% of participants lived in a household with at least one ITN; however, only 57.7% reported sleeping under the ITN. 1.5% of participants were RDT positive. Only self-reported fever was significantly correlated with RDT positivity. Conclusions With declining malaria prevalence, few individual-level characteristics were correlated with RDT positivity. This lack of correlation with individual characteristics hampers identification of individuals infected with malaria. Strategies based on ecological or environmental risk factors may be needed to target control efforts and achieve further reductions and elimination.

Abstract Background The burden of malaria has decreased dramatically within the past several years in parts of sub-Saharan Africa. Further malaria control will require targeted control strategies based on evidence of risk. The objective of this study was to identify environmental risk factors for malaria transmission using remote sensing technologies to guide malaria control interventions in a region of declining burden of malaria. Methods Satellite images were used to construct a sampling frame for the random selection of households enrolled in prospective longitudinal and cross-sectional surveys of malaria parasitaemia in Southern Province, Zambia. A digital elevation model (DEM) was derived from the Shuttle Radar Topography Mission version 3 DEM and used for landscape characterization, including landforms, elevation, aspect, slope, topographic wetness, topographic position index and hydrological models of stream networks. Results A total of 768 individuals from 128 randomly selected households were enrolled over 21 months, from the end of the rainy season in April 2007 through December 2008. Of the 768 individuals tested, 117 (15.2%) were positive by malaria rapid diagnostic test (RDT). Individuals residing within 3.75 km of a third order stream were at increased risk of malaria. Households at elevations above the baseline elevation for the region were at decreasing risk of having RDT-positive residents. Households where new infections occurred were overlaid on a risk map of RDT positive households and incident infections were more likely to be located in high-risk areas derived from prevalence data. Based on the spatial risk map, targeting households in the top 80th percentile of malaria risk would require malaria control interventions directed to only 24% of the households. Conclusions Remote sensing technologies can be used to target malaria control interventions in a region of declining malaria transmission in southern Zambia, enabling a more efficient use of resources for malaria elimination.

Abstract Background In formerly endemic areas where malaria transmission has declined, levels of population immunity to Plasmodium falciparum provide information on continued malaria transmission and potentially susceptible populations. Traditional techniques for measuring serological responses to P. falciparum antigens use plasma or dried blood spots (DBS). These invasive procedures pose a biohazard and may be unacceptable to communities if performed frequently. The use of oral fluid (OF) samples to detect antibodies to P. falciparum antigens may be a more acceptable strategy to monitor changes in population immunity. Methods An enzyme immunoassay was optimized to detect antibodies to whole, asexual stage P. falciparum antigens. Optical density (OD) values from paired DBS and OF samples collected as part of a community-based survey of malaria parasitaemia were compared. Results Oral fluid and dried blood spot samples were collected from 53 participants in Southern Province, Zambia. Their ages ranged from 1 to 80 years and 45% were female. A statistically significant correlation (r = 0.79; P < 0.01) was observed between OD values from OF and DBS samples. The OF assay identified all DBS-confirmed positive and negative samples, resulting in 100% sensitivity and specificity. Conclusions Oral fluid is a valid alternative specimen for monitoring changes in antibodies to P. falciparum antigens. As OF collection is often more acceptable to communities, poses less of a biohazard than blood samples and can be performed by community volunteers, serological surveys using OF samples provide a strategy for monitoring population immunity in regions of declining malaria transmission.

Abstract Background Asymptomatic reservoirs of malaria parasites are common yet are difficult to detect, posing a problem for malaria control. If control programmes focus on mosquito control and treatment of symptomatic individuals only, malaria can quickly resurge if interventions are scaled back. Foci of parasite populations must be identified and treated. Therefore, an active case detection system that facilitates detection of asymptomatic parasitaemia and gametocyte carriers was developed and tested in the Macha region in southern Zambia. Methods Each week, nurses at participating rural health centres (RHC) communicated the number of rapid diagnostic test (RDT) positive malaria cases to a central research team. During the dry season when malaria transmission was lowest, the research team followed up each positive case reported by the RHC by a visit to the homestead. The coordinates of the location were obtained by GPS and all consenting residents completed a questionnaire and were screened for malaria using thick blood film, RDT, nested-PCR, and RT-PCR for asexual and sexual stage parasites. Persons who tested positive by RDT were treated with artemether/lumefantrine (Coartem®). Data were compared with a community-based study of randomly selected households to assess the prevalence of asymptomatic parasitaemia in the same localities in September 2009. Results In total, 186 and 141 participants residing in 23 case and 24 control homesteads, respectively, were screened. In the case homesteads for which a control population was available (10 of the 23), household members of clinically diagnosed cases had a 8.0% prevalence of malaria using PCR compared to 0.7% PCR positive individuals in the control group (p = 0.006). The case and control groups had a gametocyte prevalence of 2.3% and 0%, respectively but the difference was not significant (p = 0.145). Conclusions This pilot project showed that active case detection is feasible and can identify reservoirs of asymptomatic infection. A larger sample size, data over multiple low transmission seasons, and in areas with different transmission dynamics are needed to further validate this approach.

Background: Reactive case detection (RCD) seeks to enhance malaria surveillance and control by identifying and treating parasitaemic individuals residing near index cases. In Zambia, this strategy starts with passive detection of symptomatic incident malaria cases at local health facilities or by community health workers, with subsequent home visits to screen-and-treat residents in the index case and neighbouring (secondary) households within a 140-m radius using rapid diagnostic tests (RDTs). However, a small circular radius may not be the most efficient strategy to identify parasitaemic individuals in low-endemic areas with hotspots of malaria transmission. To evaluate if RCD efficiency could be improved by increasing the probability of identifying parasitaemic residents, environmental risk factors and a larger screening radius (250 m) were assessed in a region of low malaria endemicity. Methods: Between January 12, 2015 and July 26, 2017, 4170 individuals residing in 158 index and 531 secondary households were enrolled and completed a baseline questionnaire in the catchment area of Macha Hospital in Choma District, Southern Province, Zambia. Plasmodium falciparum prevalence was measured using PfHRP2 RDTs and quantitative PCR (qPCR). A Quickbird™ high-resolution satellite image of the catchment area was used to create environmental risk factors in ArcGIS, and generalized estimating equations were used to evaluate associations between risk factors and secondary households with parasitaemic individuals. Results: The parasite prevalence in secondary (non-index case) households was 0.7% by RDT and 1.8% by qPCR. Overall, 8.5% (n = 45) of secondary households had at least one resident with parasitaemia by qPCR or RDT. The risk of a secondary household having a parasitaemic resident was significantly increased in proximity to higher order streams and marginally with increasing distance from index households. The adjusted OR for proximity to third- and fifth-order streams were 2.97 (95% CI 1.04–8.42) and 2.30 (95% CI 1.04–5.09), respectively, and that for distance to index households for each 50 m was 1.24 (95% CI 0.98–1.58). Conclusion: Applying proximity to streams as a screening tool, 16% (n = 3) more malaria-positive secondary households were identified compared to using a 140-m circular screening radius. This analysis highlights the potential use of environmental risk factors as a screening strategy to increase RCD efficiency. [ABSTRACT FROM AUTHOR]

Background: Insecticide-treated nets (ITNs) reduce malaria morbidity and mortality in endemic areas. Despite increasing availability, the use of ITNs remains limited in some settings. Poor malaria knowledge is a barrier to the widespread use of ITNs. The goal of this study was to assess the levels of malaria knowledge and evaluate factors associated with bed net use among individuals residing in three regions of southern Africa with different levels of malaria transmission and control. Methods: A cross-sectional study was conducted on a sample of 7535 residents recruited from 2066 households in Mutasa District, Zimbabwe (seasonal malaria transmission), Choma District, Zambia (low transmission) and Nchelenge District, Zambia (high transmission), between March 2012 and March 2017. A standardized questionnaire was used to collect data on demographics, malaria-related knowledge and use of preventive measures. Multivariate logistic regression analyses were used to assess determinants of bed net use. Results: Most of the 3836 adult participants correctly linked mosquito bites to malaria (85.0%), mentioned at least one malaria symptom (95.5%) and knew of the benefit of sleeping under an ITN. Bed net ownership and use were highest in Choma and Nchelenge Districts and lowest in Mutasa District. In multivariate analyses, knowledge of ITNs was associated with a 30-40% increased likelihood of bed net use after adjusting for potential confounders across all sites. Other factors significantly associated with bed net use were age, household size and socioeconomic status, although the direction, strength and size of association varied by study site. Importantly, participants aged 5-14 years had reduced odds of sleeping under a bed net compared to children younger than 5 years. Conclusion: Relevant knowledge of ITNs translated into the expected preventive behaviour of sleeping under a bed net, underscoring the need for continued health messaging on malaria prevention. The implementation and delivery of malaria control and elimination interventions needs to consider socioeconomic equity gaps, and target school-age children to ensure access to and improve utilization of ITNs. [ABSTRACT FROM AUTHOR]

Background: As malaria transmission declines in many regions of sub-Saharan Africa, interventions to identify the asymptomatic reservoir are being deployed with the goals of improving surveillance and interrupting transmission. Reactive case detection strategies, in which individuals with clinical malaria are followed up at their home and household residents and neighbours are screened and treated for malaria, are increasingly used as part of malaria elimination programmes. Methods: A reactive screen-and-treat programme was implemented by the National Malaria Control Centre in Southern Province, Zambia, in which individuals residing within 140 m of an index case were screened with a malaria rapid diagnostic test (RDT) and treated if positive. The operational challenges during the early stages of implementing this reactive screen-and-treat programme in the catchment area of Macha Hospital in Southern Province, Zambia were assessed using rural health centre records, ground truth evaluation of community health worker performance, and data from serial cross-sectional surveys. The proportion of individuals infected with Plasmodium falciparum who were identified and treated was estimated by simulating reactive screen-and-treat and focal drug administration cascades. Results: Within the 1st year of implementation, community health workers followed up 32 % of eligible index cases. When index cases were followed up, 66 % of residents were at home in the index households and 58 % in neighbouring households. Forty-one neighbouring households of 26 index households were screened, but only 13 (32 %) were within the 140-m screening radius. The parasite prevalence by RDT was 22 % in index households and 5 % in neighbouring households. In a simulation model with complete follow-up, 22 % of the total infected population would be detected with reactive screen-and-treat but 57 % with reactive focal drug administration. Conclusions: With limited resources, coverage and diagnostic tools, reactive screen-and-treat will likely not be sufficient to achieve malaria elimination in this setting. However, high coverage with reactive focal drug administration could be efficient at decreasing the reservoir of infection and should be considered as an alternative strategy. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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.

Malaria elimination strategies are designed to more effectively identify and treat infected individuals to interrupt transmission. One strategy, reactive screen-and-treat, starts with passive detection of symptomatic cases at health facilities. Individuals residing within the index case and neighboring households are screened with a malaria rapid diagnostic test (RDT) and treated if positive. However, it is unclear to what extent this strategy is effective in reducing transmission. Reactive screen-and-treat was implemented in Choma district, Southern Province, Zambia, in 2013, in which residents of the index case and neighboring households within 140 m were screened with an RDT. From March 2016 to July 2018, the screening radius was extended to 250-m, and additional follow-up visits at 30 and 90 days were added to evaluate the strategy. Plasmodium falciparum parasite prevalence was measured using an RDT and by quantitative PCR (qPCR). A 24-single nucleotide polymorphism molecular bar-code assay was used to genotype parasites. Eighty-four index case households with 676 residents were enrolled between March 2016 and March 2018. Within each season, parasite prevalence declined significantly in index households at the 30-day visit and remained low at the 90-day visit. However, parasite prevalence was not reduced to zero. Infections identified by qPCR persisted between study visits and were not identified by RDT. Parasites identified within the same household were most genetically related; however, overall parasite relatedness was low and similar across time and space. Thus, despite implementation of a reactive screen-and-treat program, parasitemia was not eliminated, and persisted in targeted households for at least 3 months.

To achieve and sustain malaria elimination, identification and treatment of the asymptomatic infectious reservoir is critical. Malaria rapid diagnostic tests (RDTs) are frequently used to identify asymptomatic, Plasmodium -infected individuals through test-and-treat strategies, but their sensitivity is low when used in low transmission settings. Characteristics of individuals with subpatent (RDT-negative but polymerase chain reaction [PCR]-positive) Plasmodium parasitemia were evaluated in southern Zambia where malaria transmission has declined and efforts to achieve malaria elimination are underway. Simple random sampling based on satellite imagery was used to select households for participation in community-based, cross-sectional surveys between 2008 and 2013. Questionnaires were administered to collect information on age, gender, recent history of malaria symptoms, and recent antimalarial drug use. Blood samples were collected by finger prick for Plasmodium falciparum histidine-rich protein 2 RDT, blood smears for microscopy, and dried blood spots for molecular analysis to detect malaria parasites and their sexual stage. Of 3,863 participants with complete data, 102 (2.6%) were positive by microscopy, RDT, or PCR. Of these, 48 (47%) had subpatent parasitemia. Most individuals with subpatent parasitemia were asymptomatic (85%). Compared with individuals without parasitemia, individuals with subpatent parasitemia were significantly more likely to be aged 5-25 years. Approximately one quarter (27%) of those with subpatent parasitemia had detectable gametocytemia. These findings suggest that strategies based on active or reactive case detection can identify asymptomatic individuals positive by RDT, but more sensitive diagnostic tests or focal drug administration may be necessary to target individuals with subpatent parasitemia to achieve malaria elimination.