Land use changes disrupt ecosystems, altering the transmission of vector-borne diseases. These changes have been associated with increasing incidence of zoonotic malaria caused by Plasmodium knowlesi; however, the population-level distributions of infection and exposure remain unknown. We aimed to measure prevalence of serological exposure to P knowlesi and assess associated risk factors. We did an environmentally stratified, population-based, cross-sectional survey across households in the Kudat, Kota Marudu, Pitas, and Ranau districts in northern Sabah, Malaysia, encompassing a range of ecologies. Using blood samples, the transmission intensity of P knowlesi and other malaria species was measured by specific antibody prevalence and infection detected using molecular methods. Proportions and configurations of land types were extracted from maps derived from satellite images; a data-mining approach was used to select variables. A Bayesian hierarchical model for P knowlesi seropositivity was developed, incorporating questionnaire data about individual and household-level risk factors with selected landscape factors. Between Sept 17, 2015, and Dec 12, 2015, 10 100 individuals with a median age of 25 years (range 3 months to 105 years) were sampled from 2849 households in 180 villages. 5·1% (95% CI 4·8-5·4) were seropositive for P knowlesi, and marked historical decreases were observed in the transmission of Plasmodium falciparum and Plasmodium vivax. Nine Plasmodium spp infections were detected. Age, male sex, contact with macaques, forest use, and raised house construction were positively associated with P knowlesi exposure, whereas residing at higher geographical elevations and use of insecticide were protective. Agricultural and forest variables, such as proportions and fragmentation of land cover types, predicted exposure at different spatial scales from households. Although few infections were detected, P knowlesi exposure was observed in all demographic groups and was associated with occupational factors. Results suggest that agricultural expansion and forest fragmentation affect P knowlesi exposure, supporting linkages between land use change and P knowlesi transmission. UK Medical Research Council, Natural Environment Research Council, Economic and Social Research Council, and Biotechnology and Biosciences Research Council.

Motivated by the success in malaria control that was documented over the last decade Ethiopia is aiming at malaria elimination by 2020 in selected districts. It is currently unknown if asymptomatic, submicroscopic malaria parasite carriage may form a hurdle to achieve elimination. The elimination effort may further be complicated by possible glucose-6 phosphate dehydrogenase (G6PD) deficiency which would hinder the use of 8-aminoquinolines in the elimination efforts. In February 2014 a community-based cross-sectional survey was conducted in Malo, southwest Ethiopia. Finger-prick blood samples (n = 555) were tested for presence of Plasmodium falciparum and Plasmodium vivax with microscopy, rapid diagnostic test (RDT), and nested polymerase chain reaction (nPCR). Multiplicity of P. falciparum infections was determined based on genotyping the polymorphic merozoite surface protein-2 (MSP-2) gene. Individuals were also genotyped for mutations in the gene that produces G6PD. All study participants were malaria infection negative by microscopy and RDT. Nested PCR revealed P. falciparum mono-infection in 5.2% (29/555), P. vivax mono-infection in 4.3% (24/555) and mixed infection in 0.2% (1/555) of individuals. All parasitemic individuals were afebrile (axillary temperature <37.5°C). None of the study participants carried mutations for the G6PD African A-(202GA) and Mediterranean (563CT) variants. All infections, except one, were single-clone infection by MSP-2 genotyping. The detection of a substantial number of subpatent malaria infections in an apparently asymptomatic population without evidence for malaria transmission by conventional diagnostics raises questions about the path to malaria elimination. It is currently unknown how important these infections are for sustaining malaria transmission in the study sites. The absence of G6PD deficiency indicates that 8-aminoquinolines may be safely deployed to accelerate elimination initiatives.

Background: Increasingly, vaccine efficacy studies are being recommended in low-and-middle-income countries (LMIC), yet often facilities are unavailable to take and store infant blood samples correctly. Dried blood spots (DBS), are useful for collecting blood from infants for diagnostic purposes, especially in low-income settings, as the amount of blood required is miniscule and no refrigeration is required. Little is known about their utility for antibody studies in children. This systematic review aims to investigate the correlation of antibody concentrations against infectious diseases in DBS in comparison to serum or plasma samples that might inform their use in vaccine clinical trials.Methods and findings: We searched MEDLINE, Embase and the Cochrane library for relevant studies between January 1990 to October 2020 with no language restriction, using PRISMA guidelines, investigating the correlation between antibody concentrations in DBS and serum or plasma samples, and the effect of storage temperature on DBS diagnostic performance.We included 40 studies in this systematic review. The antibody concentration in DBS and serum/plasma samples reported a good pooled correlation, (r²= 0.86 (ranged 0.43 to 1.00)). Ten studies described a decline of antibody after 28 days at room temperature compared to optimal storage at -20°C, where antibodies were stable for up to 200 days. There were only five studies of anti-bacterial antibodies.Conclusions: There is a good correlation between antibody concentrations in DBS and serum/plasma samples, supporting the wider use of DBS in vaccine and sero-epidemiological studies, but there is limited data on anti-bacterial antibodies. The correct storage of DBS is critical and may be a consideration for longer term storage.

The spread of artemisinin-resistant Plasmodium falciparum is a global health concern. Myanmar stands at the frontier of artemisinin-resistant P. falciparum. Myanmar also has the highest reported malaria burden in Southeast Asia; it is integral in the World Health Organization’s plan to eliminate malaria in Southeast Asia, yet few epidemiological data exist for the general population in Myanmar. This cross-sectional, probability household survey was conducted in Phyu township, Bago Region (central Myanmar), during the wet season of 2013. Interviewers collected clinical and behavioural data, recorded tympanic temperature and obtained dried blood spots for malaria PCR and serology. Plasmodium falciparum positive samples were tested for genetic mutations in the K13 region that may confer artemisinin resistance. Estimated type-specific malaria PCR prevalence and seroprevalence were calculated, with regression analysis to identify risk factors for seropositivity to P. falciparum. Data were weighted to account for unequal selection probabilities. 1638 participants were sampled (500 households). Weighted PCR prevalence was low (n = 41, 2.5%) and most cases were afebrile (93%). Plasmodium falciparum was the most common species (n = 19. 1.1%) and five (26%) P. falciparum samples harboured K13 mutations. Plasmodium knowlesi was detected in 1.0% (n = 16) and Plasmodium vivax was detected in 0.4% (n = 7). Seroprevalence was 9.4% for P. falciparum and 3.1% for P. vivax. Seroconversion to P. falciparum was 0.003/year in the whole population, but 16-fold higher in men over 23 years old (LR test p = 0.016). This is the first population-based seroprevalence study from central Myanmar. Low overall prevalence was discovered. However, these data suggest endemic transmission continues, probably associated with behavioural risk factors amongst working-age men. Genetic mutations associated with P. falciparum artemisinin resistance, the presence of P. knowlesi and discrete demographic risk groups present opportunities and challenges for malaria control. Responses targeted to working-age men, capable of detecting sub-clinical infections, and considering all species will facilitate malaria elimination in this setting.

Background: Antibody responses have been used to characterise transmission and exposure history in malaria-endemic settings for over a decade. Such studies have typically been conducted on well-standardised enzyme-linked immunosorbent assays (ELISAs). However, recently developed quantitative suspension array technologies (qSAT) are now capable of high-throughput and multiplexed screening of up to hundreds of analytes at a time. This study presents a customised protocol for the Luminex MAGPIX© qSAT using a diverse set of malaria antigens. The aim is to develop a standardised assay for routine serological surveillance that is implementable across laboratories and epidemiological settings. Methods: A panel of eight Plasmodium falciparum recombinant antigens, associated with long- and short-lived antibody responses, was designed for the Luminex MAGPIX© platform. The assay was optimised for key steps in the protocol: antigen-bead coupling concentration, buffer composition, serum sample dilution, and bead storage conditions. Quality control procedures and data normalisation methods were developed to address high-throughput assay processing. Antigen-specific limits of quantification (LOQs) were also estimated using both in-house and WHO reference serum as positive controls. Results: Antigen-specific bead coupling was optimised across five serum dilutions and two positive controls, resulting in concentrations operational within stable analytical ranges. Coupled beads were stable after storage at room temperature (22⁰C) for up to eight weeks. High sensitivity and specificity for distinguishing positive and negative controls at serum sample dilutions of 1:500 (AUC 0.94 95%CI 0.91-0.96) and 1:1000 (AUC 0.96 95%CI 0.94-0.98) were observed. LOQs were also successfully estimated for all analytes but varied by antigen and positive control. Conclusions: This study demonstrates that developing a standardised malaria-specific qSAT protocol for a diverse set of antigens is achievable, though further optimisations may be required. Quality control and data standardisation methods may also be useful for future analysis of large sero-epidemiological surveys.

Background: Antibody responses have been used to characterise transmission and exposure history in malaria-endemic settings for over a decade. Such studies have typically been conducted on well-standardised enzyme-linked immunosorbent assays (ELISAs). However, recently developed quantitative suspension array technologies (qSAT) are now capable of high-throughput and multiplexed screening of up to hundreds of analytes at a time. This study presents a customised protocol for the Luminex MAGPIX© qSAT using a diverse set of malaria antigens. The aim is to develop a standardised assay for routine serological surveillance that is implementable across laboratories and epidemiological settings. Methods: A panel of eight Plasmodium falciparum recombinant antigens, associated with long- and short-lived antibody responses, was designed for the Luminex MAGPIX© platform. The assay was optimised for key steps in the protocol: antigen-bead coupling concentration, buffer composition, serum sample dilution, and bead storage conditions. Quality control procedures and data normalisation methods were developed to address high-throughput assay processing. Antigen-specific limits of quantification (LOQs) were also estimated using both in-house and WHO reference serum as positive controls. Results: Antigen-specific bead coupling was optimised across five serum dilutions and two positive controls, resulting in concentrations operational within stable analytical ranges. Coupled beads were stable after storage at room temperature (22⁰C) for up to eight weeks. High sensitivity and specificity for distinguishing positive and negative controls at serum sample dilutions of 1:500 (AUC 0.94 95%CI 0.91-0.96) and 1:1000 (AUC 0.96 95%CI 0.94-0.98) were observed. LOQs were also successfully estimated for all analytes but varied by antigen and positive control. Conclusions: This study demonstrates that developing a standardised malaria-specific qSAT protocol for a diverse set of antigens is achievable, though further optimisations may be required. Quality control and data standardisation methods may also be useful for future analysis of large sero-epidemiological surveys.