A framework for evaluating health system surveillance sensitivity to support public health decision-making for malaria elimination: a case study from Indonesia.

BACKGROUND: The effectiveness of a surveillance system to detect infections in the population is paramount when confirming elimination. Estimating the sensitivity of a surveillance system requires identifying key steps in the care-seeking cascade, from initial infection to confirmed diagnosis, and quantifying the probability of appropriate action at each stage. Using malaria as an example, a framework was developed to estimate the sensitivity of key components of the malaria surveillance cascade. METHODS: Parameters to quantify the sensitivity of the surveillance system were derived from monthly malaria case data over a period of 36 months and semi-quantitative surveys in 46 health facilities on Java Island, Indonesia. Parameters were informed by the collected empirical data and estimated by modelling the flow of an infected individual through the system using a Bayesian framework. A model-driven health system survey was designed to collect empirical data to inform parameter estimates in the surveillance cascade. RESULTS: Heterogeneity across health facilities was observed in the estimated probability of care-seeking (range = 0.01-0.21, mean ± sd = 0.09 ± 0.05) and testing for malaria (range = 0.00-1.00, mean ± sd = 0.16 ± 0.29). Care-seeking was higher at facilities regularly providing antimalarial drugs (Odds Ratio [OR] = 2.98, 95% Credible Intervals [CI]: 1.54-3.16). Predictably, the availability of functioning microscopy equipment was associated with increased odds of being tested for malaria (OR = 7.33, 95% CI = 20.61). CONCLUSIONS: The methods for estimating facility-level malaria surveillance sensitivity presented here can help provide a benchmark for what constitutes a strong system. The proposed approach also enables programs to identify components of the health system that can be improved to strengthen surveillance and support public-health decision-making.

Plasmodium falciparum gametocyte production correlates with genetic markers of parasite replication but is not influenced by experimental exposure to mosquito biting.

BACKGROUND: Plasmodium blood-stage parasites balance asexual multiplication with gametocyte development. Few studies link these dynamics with parasite genetic markers in vivo; even fewer in longitudinally monitored infections. Environmental influences on gametocyte formation, such as mosquito exposure, may influence the parasite's investment in gametocyte production. METHODS: We investigated gametocyte production and asexual multiplication in two Plasmodium falciparum infected populations; a controlled human malaria infection (CHMI) study and a 28-day observational study in naturally infected individuals in Burkina Faso with controlled mosquito exposure. We measured gene transcript levels previously related to gametocyte formation (ap2-g, surfin1.2, surfin13.1, gexp-2) or inhibition of asexual multiplication (sir2a) and compared transcript levels to ring-stage parasite and mature gametocyte densities. FINDINGS: Three of the five markers (ap2-g, surfin1.2, surfin13.1) predicted peak gametocytaemia in the CHMI study. An increase in all five markers in natural infections was associated with an increase in mature gametocytes 14 days later; the effect of sir2a on future gametocytes was strongest (fold change = 1.65, IQR = 1.22-2.24, P = 0.004). Mosquito exposure was not associated with markers of gametocyte formation (ap2-g P = 0.277; sir2a P = 0.499) or carriage of mature gametocytes (P = 0.379). INTERPRETATION: All five parasite genetic markers predicted gametocyte formation over a single cycle of gametocyte formation and maturation in vivo; sir2a and ap2-g were most closely associated with gametocyte growth dynamics. We observed no evidence to support the hypothesis that exposure to Anopheles mosquito bites stimulates gametocyte formation. FUNDING: This work was funded by the Bill & Melinda Gates Foundation (INDIE OPP1173572), the European Research Council fellowship (ERC-CoG 864180) and UKRI Medical Research Council (MR/T016272/1) and Wellcome Center (218676/Z/19/Z).

Plasmodium knowlesi infection is associated with elevated circulating biomarkers of brain injury and endothelial activation.

Introduction: Malaria remains a major public health concern with substantial morbidity and mortality worldwide. In Malaysia, the emergence of Plasmodium knowlesi has led to a surge in zoonotic malaria cases and deaths in recent years. Signs of cerebral involvement have been observed in a non-comatose, fatal case of severe knowlesi infection, but the potential impact of this malaria species on the brain remains underexplored. To address this gap, we investigated circulating levels of brain injury, inflammation, and vascular biomarkers in a cohort of knowlesi-infected patients and controls. Methods: Archived plasma samples from 19 patients with confirmed symptomatic knowlesi infection and 19 healthy, age-matched controls from Peninsular Malaysia were analysed. A total of 52 plasma biomarkers of brain injury, inflammation, and vascular activation were measured using Luminex and SIMOA assays. Wilcoxon tests were used to examine group differences, and biomarker profiles were explored through hierarchical clustering heatmap analysis. Results: Bonferroni-corrected analyses revealed significantly elevated brain injury biomarker levels in knowlesi-infected patients, including S100B (p<0.0001), Tau (p=0.0007), UCH-L1 (p<0.0001), αSyn (p<0.0001), Park7 (p=0.0006), NRGN (p=0.0022), and TDP-43 (p=0.005). Compared to controls, levels were lower in the infected group for BDNF (p<0.0001), CaBD (p<0.0001), CNTN1 (p<0.0001), NCAM-1 (p<0.0001), GFAP (p=0.0013), and KLK6 (p=0.0126). Hierarchical clustering revealed distinct group profiles for circulating levels of brain injury and vascular activation biomarkers. Conclusions: Our findings highlight for the first time the impact of Plasmodium knowlesi infection on the brain, with distinct alterations in cerebral injury and endothelial activation biomarker profiles compared to healthy controls. Further studies are warranted to investigate the pathophysiology and clinical significance of these altered surrogate markers, through both neuroimaging and long-term neurocognitive assessments.

Malaria seroepidemiology in very low transmission settings in the Peruvian Amazon.

Despite progress towards malaria reduction in Peru, measuring exposure in low transmission areas is crucial for achieving elimination. This study focuses on two very low transmission areas in Loreto (Peruvian Amazon) and aims to determine the relationship between malaria exposure and proximity to health facilities. Individual data was collected from 38 villages in Indiana and Belen, including geo-referenced households and blood samples for microscopy, PCR and serological analysis. A segmented linear regression model identified significant changes in seropositivity trends among different age groups. Local Getis-Ord Gi* statistic revealed clusters of households with high (hotspots) or low (coldspots) seropositivity rates. Findings from 4000 individuals showed a seropositivity level of 2.5% (95%CI: 2.0%-3.0%) for P. falciparum and 7.8% (95%CI: 7.0%-8.7%) for P. vivax, indicating recent or historical exposure. The segmented regression showed exposure reductions in the 40-50 age group (ß1 = 0.043, p = 0.003) for P. vivax and the 50-60 age group (ß1 = 0.005, p = 0.010) for P. falciparum. Long and extreme distance villages from Regional Hospital of Loreto exhibited higher malaria exposure compared to proximate and medium distance villages (p < 0.001). This study showed the seropositivity of malaria in two very low transmission areas and confirmed the spatial pattern of hotspots as villages become more distant.

Characterizing the spatial distribution of multiple malaria diagnostic endpoints in a low-transmission setting in Lao PDR.

The epidemiology of malaria changes as prevalence falls in low-transmission settings, with remaining infections becoming more difficult to detect and diagnose. At this stage active surveillance is critical to detect residual hotspots of transmission. However, diagnostic tools used in active surveillance generally only detect concurrent infections, and surveys may benefit from sensitive tools such as serological assays. Serology can be used to interrogate and characterize individuals' previous exposure to malaria over longer durations, providing information essential to the detection of remaining foci of infection. We ran blood samples collected from a 2016 population-based survey in the low-transmission setting of northern Lao PDR on a multiplexed bead assay to characterize historic and recent exposures to Plasmodium falciparum and vivax. Using geostatistical methods and remote-sensing data we assessed the environmental and spatial associations with exposure, and created predictive maps of exposure within the study sites. We additionally linked the active surveillance PCR and serology data with passively collected surveillance data from health facility records. We aimed to highlight the added information which can be gained from serology as a tool in active surveillance surveys in low-transmission settings, and to identify priority areas for national surveillance programmes where malaria risk is higher. We also discuss the issues faced when linking malaria data from multiple sources using multiple diagnostic endpoints.

The acquisition of humoral immune responses targeting Plasmodium falciparum sexual stages in controlled human malaria infections.

Individuals infected with P. falciparum develop antibody responses to intra-erythrocytic gametocyte proteins and exported gametocyte proteins present on the surface of infected erythrocytes. However, there is currently limited knowledge on the immunogenicity of gametocyte antigens and the specificity of gametocyte-induced antibody responses. In this study, we assessed antibody responses in participants of two controlled human malaria infection (CHMI) studies by ELISA, multiplexed bead-based antibody assays and protein microarray. By comparing antibody responses in participants with and without gametocyte exposure, we aimed to disentangle the antibody response induced by asexual and sexual stage parasites. We showed that after a single malaria infection, a significant anti-sexual stage humoral response is induced in malaria-naïve individuals, even after exposure to relatively low gametocyte densities (up to ~1,600 gametocytes/mL). In contrast to antibody responses to well-characterised asexual blood stage antigens that were detectable by day 21 after infection, responses to sexual stage antigens (including transmission blocking vaccine candidates Pfs48/45 and Pfs230) were only apparent at 51 days after infection. We found antigens previously associated with early gametocyte or anti-gamete immunity were highly represented among responses linked with gametocyte exposure. Our data provide detailed insights on the induction and kinetics of antibody responses to gametocytes and identify novel antigens that elicit antibody responses exclusively in individuals with gametocyte exposure. Our findings provide target identification for serological assays for surveillance of the malaria infectious reservoir, and support vaccine development by describing the antibody response to leading vaccine antigens after primary infection.