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ObjectivesWe assessed the prevalence of immunoglobulin G (IgG) and immunoglobulin M (IgM) against four endemic human coronaviruses (HCoVs) and two SARS-CoV-2 antigens, among vaccinated and unvaccinated staff at health care centres in Uganda, Sierra Leone, and the Democratic Republic of Congo (DRC).MethodsGovernment health facility staff who had patient contact in Goma (DRC), Kambia District (Sierra Leone), and Masaka District (Uganda) were enrolled. Questionnaires and blood samples were collected at three timepoints over four months. Blood samples were analysed with the Luminex MAGPIX®.ResultsAmong unvaccinated participants, the prevalence of IgG/IgM antibodies against SARS-CoV-2 RBD or N-protein at enrolment was 70% in Goma (138/196), 89% in Kambia (112/126) and 89% in Masaka (190/213). IgG responses against endemic HCoVs at baseline were not associated with SAR-CoV-2 sero-acquisition during follow-up. Among vaccinated participants, those who had evidence of SARS-CoV-2 IgG/IgM at baseline tended to have higher IgG responses to vaccination compared to those SARS-CoV-2 seronegative at baseline, controlling for the time of sample collection since vaccination.ConclusionsThe high levels of natural immunity and hybrid immunity should be incorporated into both vaccination policy and prediction models of the impact of subsequent waves of infection in these settings.

BackgroundAssessing the status of malaria transmission in endemic areas becomes increasingly challenging as countries approach elimination. Serology can provide robust estimates of malaria transmission intensities, and multiplex serological assays allow for simultaneous assessment of markers of recent and historical malaria exposure.MethodsHere, we evaluated different statistical and machine learning methods for analyzing multiplex malaria-specific antibody response data to classify recent and historical exposure toPlasmodium falciparumandP. vivax. To assess these methods, we utilized samples from a health-facility based survey (n=9132) in the Philippines, where we quantified antibody responses against 8P. falciparumand 6P. vivax-specific antigens from 3 sites with varying transmission intensity.FindingsMeasurements of antibody responses and seroprevalence were consistent with the 3 sites’ known endemicity status. For predictingP. falciparuminfection, a machine learning (ML) approach (Random Forest model) using 4 serological markers (PfGLURP R2, Etramp5.Ag1, GEXP18 and PfMSP119) gave better predictions for cases in Palawan (AUC: 0·9591, CI 0·9497-0·9684) than individual antigen seropositivity. Although the ML approach did not improveP. vivaxinfection predictions, ML classifications confirmed the absence of recent exposure toP. falciparumandP. vivaxin both Occidental Mindoro and Bataan. For predicting historicalP. falciparumandP. vivaxtransmission, seroprevalence and seroconversion rates based on cumulative exposure markers AMA1 and MSP119showed reliable trends in the 3 sites.InterpretationOur study emphasizes the utility of serological markers in predicting recent and historical exposure in a sub-national elimination setting, and also highlights the potential use of machine learning models using multiplex antibody responses to improve assessment of the malaria transmission status of countries aiming for elimination. This work also provides baseline antibody data for monitoring risk in malaria-endemic areas in the Philippines.FundingNewton Fund, Philippine Council for Health Research and Development, and UK Medical Research Council.

Individuals infected with

The merozoite surface protein MSPDBL2 of Plasmodium falciparum is under strong balancing selection and is a target of naturally acquired antibodies. Remarkably, MSPDBL2 is expressed in only a minority of mature schizonts of any cultured parasite line, and mspdbl2 gene transcription increases in response to overexpression of the gametocyte development inducer GDV1, so it is important to understand its natural expression. Here, MSPDBL2 in mature schizonts was analysed in the first ex vivo culture cycle of 96 clinical isolates from four populations with varying levels of infection endemicity in different West African countries, by immunofluorescence microscopy with antibodies against a conserved region of the protein. In most isolates, less than 1% of mature schizonts were positive for MSPDBL2 (median of 0.6% overall), but the frequency distribution was highly skewed as nine isolates had more than 3% schizonts positive and one had 73% positive. To investigate whether expression of other gene loci correlated with MSPDBL2 expression, whole transcriptome sequencing was performed on schizont-enriched material from 17 of the clinical isolates with a wide range of proportions of schizonts positive. Transcripts of particular parasite genes were highly significantly positively correlated with MSPDBL2 positivity in schizonts as well as with mspdbl2 gene transcript levels, with overrepresentation of genes previously implicated as likely to be involved in gametocytogenesis, but not including the gametocytogenesis master regulator ap2g. Although MSPDBL2 is apparently not directly involved in sexual commitment, it marks a co-occurring developmental subpopulation that may be functionally distinct within blood stage infections.

Summary Background The potential effects of SARS-CoV-2 and Plasmodium falciparum co-infection on host susceptibility and pathogenesis remain unknown. We aimed to establish the prevalence of malaria and describe the clinical characteristics of SARS-CoV-2 and P falciparum co-infection in a high-burden malaria setting. Methods This was an exploratory prospective, cohort study of patients with COVID-19 who were admitted to hospital in Uganda. Patients of all ages with a PCR-confirmed diagnosis of SARS-CoV-2 infection who had provided informed consent or assent were consecutively enrolled from treatment centres in eight hospitals across the country and followed up until discharge or death. Clinical assessments and blood sampling were done at admission for all patients. Malaria diagnosis in all patients was done by rapid diagnostic tests, microscopy, and molecular methods. Previous P falciparum exposure was determined with serological responses to a panel of P falciparum antigens assessed using a multiplex bead assay. Additional evaluations included complete blood count, markers of inflammation, and serum biochemistries. The main outcome was overall prevalence of malaria infection and malaria prevalence by age (including age categories of 0–20 years, 21–40 years, 41–60 years, and >60 years). The frequency of symptoms was compared between patients with COVID-19 with P falciparum infection versus those without P falciparum infection. The frequency of comorbidities and COVID-19 clinical severity and outcomes was compared between patients with low previous exposure to P falciparum versus those with high previous exposure to P falciparum. The effect of previous exposure to P falciparum on COVID-19 clinical severity and outcomes was also assessed among patients with and those without comorbidities. Findings Of 600 people with PCR-confirmed SARS-CoV-2 infection enrolled from April 15, to Oct 30, 2020, 597 (>99%) had complete information and were included in our analyses. The majority (502 [84%] of 597) were male individuals with a median age of 36 years (IQR 28–47). Overall prevalence of P falciparum infection was 12% (95% CI 9·4–14·6; 70 of 597 participants), with highest prevalence in the age groups of 0–20 years (22%, 8·7–44·8; five of 23 patients) and older than 60 years (20%, 10·2–34·1; nine of 46 patients). Confusion (four [6%] of 70 patients vs eight [2%] of 527 patients; p=0·040) and vomiting (four [6%] of 70 patients vs five [1%] of 527 patients; p=0·014] were more frequent among patients with P falciparum infection than those without. Patients with low versus those with high previous P falciparum exposure had a increased frequency of severe or critical COVID-19 clinical presentation (16 [30%] of 53 patients vs three [5%] of 56 patients; p=0·0010) and a higher burden of comorbidities, including diabetes (12 [23%] of 53 patients vs two [4%] of 56 patients; p=0·0010) and heart disease (seven [13%] of 53 patients vs zero [0%] of 56 patients; p=0·0030). Among patients with no comorbidities, those with low previous P falciparum exposure still had a higher proportion of cases of severe or critical COVID-19 than did those with high P falciparum exposure (six [18%] of 33 patients vs one [2%] of 49 patients; p=0·015). Multivariate analysis showed higher odds of unfavourable outcomes in patients who were older than 60 years (adjusted OR 8·7, 95% CI 1·0–75·5; p=0·049). Interpretation Although patients with COVID-19 with P falciparum co-infection had a higher frequency of confusion and vomiting, co-infection did not seem deleterious. The association between low previous malaria exposure and severe or critical COVID-19 and other adverse outcomes will require further study. These preliminary descriptive observations highlight the importance of understanding the potential clinical and therapeutic implications of overlapping co-infections. Funding Malaria Consortium (USA).

Due to challenges in measuring changes in malaria in low transmission settings, serology is increasingly being used to complement clinical and parasitological surveillance. Longitudinal cohort studies have shown serological markers, such as Etramp5.Ag1, to be particularly discriminatory of spatio-temporal differences in malaria transmission. However, these markers have yet to be used as endpoints in intervention trials. This study is an extended analysis of a 2017 cluster randomised trial conducted in Zambezi Region, Namibia, evaluating the effectiveness of reactive focal mass drug administration (rfMDA) and reactive vector control (RAVC). A panel of eight serological markers of Plasmodium falciparum infection - Etramp5.Ag1, GEXP18, HSP40.Ag1, Rh2.2030, EBA175, PfMSP119, PfAMA1, and PfGLURP.R2 - was used on a multiplex immunoassay to measure population antibody responses as trial endpoints.Reductions in sero-prevalence to antigens Etramp.Ag1, PfMSP119, Rh2.2030, and PfAMA1 were observed in study arms combining rfMDA and RAVC, but only effects for Etramp5.Ag1 were statistically significant. Etramp5.Ag1 sero-prevalence was significantly lower in all intervention arms. Compared to the reference arms, adjusted Etramp5.Ag1 prevalence ratio (aPR) was 0.77 (95%CI 0.65 – 0.90, pThe use of serological endpoints to evaluate trial outcomes was comparable to qPCR and measured effect size with improved precision. Serology has clear application in cluster randomised trials, particularly in settings where measuring clinical incidence or infection is less reliable due to seasonal fluctuations, limitations in health care seeking, or incomplete testing and reporting.Key questionsWhat is already known?▪Numerous serological studies across sub-Saharan Africa have found that malaria-specific antibody responses are highly correlated with malaria transmission.▪Serology is increasingly being used to complement traditional malaria surveillance data in settings where clinical or parasitological measures of incidence or infection may be less reliable due to fluctuations in parasite densities, limitations in health care seeking, or incomplete testing and reporting.▪The identification of new serological markers associated with recent malaria exposure hold promise as measures of malaria incidence. In previous longitudinal cohort studies, Etramp5.Ag1 has been shown to be a discriminatory serological marker capable of detecting spatio-temporal differences in malaria transmission. However, these markers have never been formally used as endpoints in a malaria cluster randomised trial.What are the new findings?▪This study is the first application of serological endpoints in a malaria cluster randomised trial. Using a multiplexed immunoassay, a panel of sero-incidence markers of recent malaria exposure were used to evaluate the effectiveness of reactive focal mass drug administration (rfMDA) and reactive focal vector control (RAVC) compared to reactive case detection (standard of care) to reduce malaria transmission.▪Cluster-level antibody responses were significantly lower in all intervention arms compared to control, and effect sizes were measured with greater study power than other trial endpoints such as quantitative polymerase chain reaction (qPCR) parasite prevalence.What do the new findings imply?▪The findings from this study, together with ongoing innovations in assay design and multi-disease platforms, illustrate the potential application of serological markers as endpoints in cluster randomised trials. The use of serological endpoints can help achieve trial efficiencies, such as reduced sample size, particularly in low transmission settings or multi-intervention trials where measuring differences between study arms may be challenging with clinical or parasitological endpoints alone.

Rationale: Nasopharyngeal administration of live virulence-attenuated Streptococcus pneumoniae strains is a potential novel preventative strategy. One target for creating reduced virulence S. pneumoniae strains is the capsule, but loss of the capsule reduces the duration of S. pneumoniae colonisation in mice which could impair protective efficacy against subsequent infection. Objectives: To assess protective efficacy of nasopharyngeal administration of unencapsulated S. pneumoniae strains in murine infection models. Methods: Strains containing cps locus deletions combined with the S. pneumoniae virulence factors psaA (reduces colonisation) or proABC (no effect on colonisation) were constructed and their virulence phenotypes and ability to prevent recolonisation or invasive infection assessed using mouse infection models. Serological responses to colonisation were compared between strains using ELISAs, immunoblots and 254 S. pneumoniae protein antigen array. Measurements and Main Results: The ∆cps/piaA and ∆cps/proABC strains were strongly attenuated in virulence in both invasive infection models and had a reduced ability to colonise the nasopharynx. ELISAs, immunoblots and protein arrays showed colonisation with either strain stimulated weaker serological responses than the wild type strain. Mice previously colonised with these strains were protected against septicaemic pneumonia but, unlike mice colonised with the wild type strain, not against S. pneumoniae recolonisation. Conclusions: Colonisation with the ∆cps/piaA and ∆cps/proABC strains prevented subsequent septicaemia, but in contrast, to published data for encapsulated double mutant strains they did not prevent recolonisation with S. pneumoniae. These data suggest targeting the cps locus is a less effective option for creating live attenuated strains that prevent S. pneumoniae infections.

Within the overlapping geographical ranges of P. knowlesi monkey hosts and vectors in Southeast Asia, an estimated 1.5 billion people are considered at risk of infection. P. knowlesi can cause severe disease and death, the latter associated with delayed treatment occurring from misdiagnosis. Although microscopy is a sufficiently sensitive first-line tool for P. knowlesi detection for most low-level symptomatic infections, misdiagnosis as other Plasmodium species is common, and the majority of asymptomatic infections remain undetected. Current point-of-care rapid diagnostic tests demonstrate insufficient sensitivity and poor specificity for differentiating P. knowlesi from other Plasmodium species. Molecular tools including nested, real-time, and single-step PCR, and loop-mediated isothermal amplification (LAMP), are sensitive for P. knowlesi detection. However, higher cost and inability to provide the timely point-of-care diagnosis needed to guide appropriate clinical management has limited their routine use in most endemic clinical settings. P. knowlesi is likely underdiagnosed across the region, and improved diagnostic and surveillance tools are required. Reference laboratory molecular testing of malaria cases for both zoonotic and non-zoonotic Plasmodium species needs to be more widely implemented by National Malaria Control Programs across Southeast Asia to accurately identify the burden of zoonotic malaria and more precisely monitor the success of human-only malaria elimination programs. The implementation of specific serological tools for P. knowlesi would assist in determining the prevalence and distribution of asymptomatic and submicroscopic infections, the absence of transmission in certain areas, and associations with underlying land use change for future spatially targeted interventions.

Background: Our understanding of the potential impact of SARS-CoV-2 and malaria co-infection on host susceptibility and pathogenesis remains unclear. We determined the prevalence of malaria and describe the consequences of SARS-CoV-2 and malaria co-infection in a high burden malaria setting. Methods: This was a prospective cohort study of hospitalized Covid-19 patients in Uganda. Malaria diagnosis was done using rapid diagnostic tests, microcopy and molecular methods. Previous P. falciparum exposure was assessed using serologic responses to a panel of P. falciparum antigens using a multiplex bead assay. Additional evaluations included complete blood count, markers of inflammation and serum biochemistries. Findings: Of 597 PCR confirmed Covid-19 cases enrolled between 16th April and 30th October 2020, 500 (84.1%) were male and median age (1QR) was 36 (28-47) years. Overall prevalence of P. falciparum infection was 11.7% (70/597, 95% CI 9.4 to 14.6), with highest prevalence in the 0- 20 years (21.7%, 5/23, 95% CI 8.7-44.8) and > 60 years (19.6%, 9/46, 95% CI 10.2-34.1) age groups. Confusion [5.7% (4/70) vs. 1.5%, (8/527), p=0.04] and vomiting [5.7% (5/70) vs.1.0%, 5/527), p=0.007] were more frequent among patients with P. falciparum infection. Patients with low previous P. falciparum exposure had a higher frequency of severe/critical Covid-19 cases (30.2%, 16/53, p=0.001), a higher burden of comorbidities [hypertension (30.2%, 16/53, p=0.02) and diabetes (22.6%, 12/53, p=0.003)] and more deaths (3.8%, 2/53, p=0.01). Among patients with no comorbidities, those with low previous exposure still had a higher proportion of severe/critical Covid-19 cases (18.2%, 6/53 vs. 2.0%, 1/56, p=0.01) compared to those with high exposure. Interpretation: Prevalence of P. falciparum infection among Covid-19 patients was relatively high. Though Covid-19 patients with P. falciparum infection had a higher frequency of confusion and vomiting, co-infection with malaria did not seem deleterious. Low previous malaria exposure was associated with severe/critical Covid-19 and adverse outcomes. Funding: Funding: Grant from Malaria Consortium, US Declaration of Interest: HA and FN are members of the Mulago Hospital Research and Ethics but did not participate in decisions pertaining this study. All other authors declare no competing interests. Ethical Approval: The study was approved by the Mulago National Referral Hospital Research and Ethics committee and the Uganda National Council for Science and Technology.

The evaluation of protein antigens as putative serologic biomarkers of infection has increasingly shifted to high-throughput, multiplex approaches such as the protein microarray. In vitro transcription/translation (IVTT) systems – a similarly high-throughput protein expression method – are already widely utilised in the production of protein microarrays, though purified recombinant proteins derived from more traditional whole cell based expression systems also play an important role in biomarker characterisation. Here we have performed a side-by-side comparison of antigen-matched protein targets from an IVTT and purified recombinant system, on the same protein microarray. The magnitude and range of antibody responses to purified recombinants was found to be greater than that of IVTT proteins, and responses between targets from different expression systems did not clearly correlate. However, responses between amino acid sequence-matched targets from each expression system were more closely correlated. Despite the lack of a clearly defined relationship between antigen-matched targets produced in each expression system, our data indicate that protein microarrays produced using either method can be used confidently, in a context dependent manner, though care should be taken when comparing data derived from contrasting approaches.Statement of significance of the studyProtein microarray technology is increasingly being realised as a powerful tool in disease biomarker identification. Protein-based, serologic arrays are already well utilised in the characterisation of antibody responses to the malarial Plasmodium spp., drastically improving throughput and efficiency compared to more typical experimental assays. Such approaches have commonly made use of in vitro transcription/translation (IVTT) protein expression systems, though other protein expression methods are regularly employed. In this study we have directly compared antibody responses in a malaria endemic population, to matched protein antigens derived from both an IVTT and purified bacterial recombinant system in the same protein microarray. We demonstrated that the magnitude of measured antibody response to matched protein antigens tends to be greater to purified recombinants, rather than IVTT products. Further, our analysis showed highly variable levels of correlation of response between antigen-matched targets derived from each expression system, although relationships in antibody response were stronger between proteins with overlapping amino acid sequences. This study highlights the importance of considering the strengths and weaknesses of each expression system in the context of a protein microarray according to experimental hypotheses, and illustrates the need for attention when comparing data generated by these different methodologies.

Chagas disease is considered the most important parasitic disease in Latin America. The protozoan agent, Trypanosoma cruzi, comprises six genetic lineages, TcI-TcVI. Genotyping to link lineage(s) to severity of cardiomyopathy and gastrointestinal pathology is impeded by the sequestration and replication of T. cruzi in host tissues. We describe serology specific for TcI, the predominant lineage north of the Amazon, based on expression of recombinant trypomastigote small surface antigen (gTSSA-I) in the eukaryote Leishmania tarentolae, to allow realistic glycosylation and structure of the antigen. Sera from TcI-endemic regions recognised gTSSA-I (74/146; 50.7%), with no cross reaction with common components of gTSSA-II/V/VI recombinant antigen. Antigenicity was abolished by chemical (periodate) oxidation of gTSSA-I glycosylation but retained after heat-denaturation of conformation. Conversely, non-specific recognition of gTSSA-I by non-endemic malaria sera was abolished by heat-denaturation. TcI-specific serology facilitates investigation between lineage and diverse clinical presentations. Glycosylation cannot be ignored in the search for immunogenic antigens.

Background As malaria transmission declines, sensitive diagnostics are needed to evaluate interventions and monitor transmission. Serological assays measuring malaria antibody responses offer a cost-effective detection method to supplement existing surveillance tools. Methods A prospective cohort study was conducted from 2013 to 2015 in 12 villages across five administrative regions in The Gambia. Serological analysis included samples from the West Coast Region at the start and end of the season (July and December 2013) and from the Upper River Region in July and December 2013 and April and December 2014. Antigen-specific antibody responses to eight Plasmodium falciparum (P. falciparum) antigens—Etramp5.Ag1, GEXP18, HSP40.Ag1, Rh2.2030, EBA175 RIII-V, PfMSP119, PfAMA1, and PfGLURP.R2—were quantified using a multiplexed bead-based assay. The association between antibody responses and clinical and parasitological endpoints was estimated at the individual, household, and population level. Results Strong associations were observed between clinical malaria and concurrent sero-positivity to Etramp5.Ag1 (aOR 4.60 95% CI 2.98–7.12), PfMSP119 (aOR 4.09 95% CI 2.60–6.44), PfAMA1 (aOR 2.32 95% CI 1.40–3.85), and PfGLURP.R2 (aOR 3.12, 95% CI 2.92–4.95), while asymptomatic infection was associated with sero-positivity to all antigens. Village-level sero-prevalence amongst children 2–10 years against Etramp5.Ag1, HSP40.Ag1, and PfMSP119 showed the highest correlations with clinical and P. falciparum infection incidence rates. For all antigens, there were increased odds of asymptomatic P. falciparum infection in subjects residing in a compound with greater than 50% sero-prevalence, with a 2- to 3-fold increase in odds of infection associated with Etramp5.Ag1, GEXP18, Rh2.2030, PfMSP119, and PfAMA1. For individuals residing in sero-positive compounds, the odds of clinical malaria were reduced, suggesting a protective effect. Conclusions At low transmission, long-lived antibody responses could indicate foci of malaria transmission that have been ongoing for several seasons or years. In settings where sub-patent infections are prevalent and fluctuate below the detection limit of polymerase chain reaction (PCR), the presence of short-lived antibodies may indicate recent infectivity, particularly in the dry season when clinical cases are rare. Serological responses may reflect a persistent reservoir of infection, warranting community-targeted interventions if individuals are not clinically apparent but have the potential to transmit. Therefore, serological surveillance at the individual and household level may be used to target interventions where there are foci of asymptomatically infected individuals, such as by measuring the magnitude of age-stratified antibody levels or identifying areas with clustering of above-average antibody responses across a diverse range of serological markers.

Background: Antimalarial antibody measurements are useful because they reflect historical and recent exposure to malaria. As such, they may provide additional information to assess ongoing transmission in low endemic or pre-elimination settings where cases are rare. In addition, the absence of antibody responses in certain individuals can indicate the cessation of transmission. Commercial malaria enzyme-linked immunosorbent assays (ELISA) detect antimalarial antibodies and are commonly used to screen blood donations for possible malaria infection. However, there is no standardized test to detect antimalarial antibodies for epidemiological use. Here we compared five commercially available ELISA kits (Trinity Biotech, newbio, DiaPro, Cellabs, and NovaTec) in search of a standardized tool for supporting claims of absence of malaria transmission. For comparison, a research-based (RB) ELISA protocol was performed alongside the commercial kits.\ud \ud Results: The commercial kits were first compared using serum samples from known malaria-unexposed individuals (n = 223) and Toxoplasma-infected individuals (n = 191) to assess specificity and cross-reactivity against non-malaria infections. In addition, 134 samples from ≥10-year-olds collected in a hyperendemic region in the Gambia in the early 1990s were used to assess sensitivity. Three out of five kits showed high sensitivity (90–92%), high specificity (98–99%), low cross-reactivity (0–3%) and were considered user-friendly (Trinity Biotech, newbio and NovaTec). Two of these kits (Trinity Biotech and NovaTec) were taken forward for epidemiological evaluation and results were compared to those using the RB-ELISA. Samples from two pre-elimination settings (Praia, Cape Verde; n = 1,396, and Bataan, the Philippines; n = 1,824) were tested. Serological results from both the Trinity Biotech kit and the RB-ELISA concurred with recent passively detected case counts in both settings. Results from the Trinity Biotech kit reflected a significant decrease in the number of reported cases in Bataan in the 1990s better than the RB-ELISA. Results from the NovaTec kit did not reflect transmission patterns in either setting.\ud \ud Conclusion: The Trinity Biotech commercial ELISA kit was considered reliable for epidemiological use and accurately described transmission patterns in two (previously) malaria endemic settings. The use of this simple and standardized serological tool may aid national control and elimination programs by confirming that regions are free from malaria.

The island of Hispaniola aims to eliminate malaria by 2025; however, there are limited data to describe epidemiologic risk factors for malaria in this setting. A prospective case–control study was conducted at four health facilities in southwest Haiti, aiming to describe factors influencing the risk of current and past malaria infection. Cases were defined as individuals attending facilities with current or recent fever and positive malaria rapid diagnostic test (RDT), while controls were those with current or recent fever and RDT negative. Serological markers of recent and cumulative exposure to Plasmodium were assessed using the multiplex bead assay from dried blood spots and used for alternate case definitions. Kuldorff’s spatial scan statistic was used to identify local clusters of infection or exposure. Logistic regression models were used to assess potential risk factors for RDT positivity and recent exposure markers, including age-group, gender, and recruiting health facility as group-matching variables. A total of 192 cases (RDT positive) and 915 controls (RDT negative) were recruited. Consistent spatial clusters were identified for all three infection and exposure metrics, indicating temporal stability of malaria transmission at these sites. Risk factors included remoteness from health facilities and household construction, furthermore, insecticide-treated net ownership or use was associated with reduced odds of RDT positivity. These findings indicate the malaria risk in Grand’Anse is driven primarily by location. Travel, occupation, and other behavioral factors were not associated with malaria. These data can support the National Malaria Program to refine and target their intervention approaches, and to move toward elimination.

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 In order to improve malaria burden estimates in low transmission settings, more sensitive tools and efficient sampling strategies are required. This study evaluated the use of serological measures from repeated health facility-based cross-sectional surveys to investigate Plasmodium falciparum and Plasmodium vivax transmission dynamics in an area nearing elimination in Indonesia. Methods Quarterly surveys were conducted in eight public health facilities in Kulon Progo District, Indonesia, from May 2017 to April 2018. Demographic data were collected from all clinic patients and their companions, with household coordinates collected using participatory mapping methods. In addition to standard microscopy tests, bead-based serological assays were performed on finger-prick bloodspot samples from 9453 people. Seroconversion rates (SCR, i.e. the proportion of people in the population who are expected to seroconvert per year) were estimated by fitting a simple reversible catalytic model to seroprevalence data. Mixed effects logistic regression was used to examine factors associated with malaria exposure, and spatial analysis was performed to identify areas with clustering of high antibody responses. Results Parasite prevalence by microscopy was extremely low (0.06% (95% confidence interval 0.03–0.14, n = 6) and 0 for P. vivax and P. falciparum, respectively). However, spatial analysis of P. vivax antibody responses identified high-risk areas that were subsequently the site of a P. vivax outbreak in August 2017 (62 cases detected through passive and reactive detection systems). These areas overlapped with P. falciparum high-risk areas and were detected in each survey. General low transmission was confirmed by the SCR estimated from a pool of the four surveys in people aged 15 years old and under (0.020 (95% confidence interval 0.017–0.024) and 0.005 (95% confidence interval 0.003–0.008) for P. vivax and P. falciparum, respectively). The SCR estimates in those over 15 years old were 0.066 (95% confidence interval 0.041–0.105) and 0.032 (95% confidence interval 0.015–0.069) for P. vivax and P. falciparum, respectively. Conclusions These findings demonstrate the potential use of health facility-based serological surveillance to better identify and target areas still receptive to malaria in an elimination setting. Further implementation research is needed to enable integration of these methods with existing surveillance systems.

Measuring antimalarial antibodies can estimate transmission in a population. To compare outputs, standardized laboratory testing is required. Here we describe the in-country establishment and quality control (QC) of a multiplex bead assay (MBA) for three sero-surveys in Haiti. Total IgG data against 21 antigens were collected for 32,758 participants. Titration curves of hyperimmune sera were included on assay plates, assay signals underwent 5-parameter regression, and inspection of the median and interquartile range (IQR) for the y-inflection point was used to determine assay precision. The medians and IQRs were similar for Surveys 1 and 2 for most antigens, while the IQRs increased for some antigens in Survey 3. Levey-Jennings charts for selected antigens provided a pass/fail criterion for each assay plate and, of 387 assay plates, 13 (3.4%) were repeated. Individual samples failed if IgG binding to the generic glutathione-S-transferase protein was observed, with 659 (2.0%) samples failing. An additional 455 (1.4%) observations failed due to low bead numbers (

Background As malaria transmission declines, sensitive diagnostics are needed to evaluate interventions and monitor transmission. Serological assays measuring malaria antibody responses offer a cost-effective detection method to supplement existing surveillance tools. Methods A prospective cohort study was conducted from 2013 to 2015 in 12 villages across five administrative regions in The Gambia. Serological analysis included samples from the West Coast Region at the start and end of the season (July and December 2013) and from the Upper River Region in July and December 2013, and April and December 2014. Antigen-specific antibody responses to seven Plasmodium falciparum ( P.falciparum ) antigens - Etramp5.Ag1, GEXP18, HSP40.Ag1, Rh2.2030, EBA175 RIII-V, Pf MSP1 19 , Pf AMA1 - were quantified using a multiplexed bead-based assay. The association between antibody responses and clinical and parasitological endpoints was estimated at the individual, household, and population level. Results Strong associations were observed between clinical malaria and concurrent sero-positivity to Etramp5.Ag1 (aOR 4.56 95%CI 2.94 -7.06), Pf MSP1 19 (aOR 3.84 95%CI 2.41 - 6.04) and Pf AMA1 (aOR 2.18 95%CI 1.23 - 3.84), while asymptomatic infection was associated with sero-positivity to all antigens. Village-level sero-prevalence amongst children 2-10 years against Etramp5.Ag1, HSP40.Ag1, and Pf MSP1 19 showed the highest correlations with clinical and P.falciparum infection incidence rates. For all antigens, there was increased odds of asymptomatic P.falciparum infection in subjects residing in a compound with greater than 50% sero-prevalence, with a 2- to 3-fold increase in odds of infection associated with Etramp5.Ag1, GEXP18, Rh2.2030, Pf MSP1 19 and Pf AMA1. For individuals residing in sero-positive compounds, the odds of clinical malaria were reduced, suggesting a protective effect. Conclusions At low transmission, long-lived antibody responses could indicate foci of malaria transmission that have been ongoing for several seasons or years. In settings where sub-patent infections are prevalent and fluctuate below the detection limit of polymerase chain reaction (PCR), the presence of short-lived antibodies may indicate recent infectivity, particularly in the dry season when clinical cases are rare. Serological responses may reflect a persistent reservoir of infection, warranting community-targeted interventions if individuals are not clinically apparent but have the potential to transmit. Therefore, serological surveillance at both the individual and household level may be used to target interventions where there are foci of asymptomatically infected individuals.

BackgroundWe assessed the impact of exposure to Plasmodium falciparum on parasite kinetics, clinical symptoms, and functional immunity after controlled human malaria infection (CHMI) in 2 cohorts with different levels of previous malarial exposure.MethodsNine adult males with high (sero-high) and 10 with low (sero-low) previous exposure received 3200 P. falciparum sporozoites (PfSPZ) of PfSPZ Challenge by direct venous inoculation and were followed for 35 days for parasitemia by thick blood smear (TBS) and quantitative polymerase chain reaction. Endpoints were time to parasitemia, adverse events, and immune responses.ResultsTen of 10 (100%) volunteers in the sero-low and 7 of 9 (77.8%) in the sero-high group developed parasitemia detected by TBS in the first 28 days (P = .125). The median time to parasitemia was significantly shorter in the sero-low group than the sero-high group (9 days [interquartile range {IQR} 7.5–11.0] vs 11.0 days [IQR 7.5–18.0], respectively; log-rank test, P = .005). Antibody recognition of sporozoites was significantly higher in the sero-high (median, 17.93 [IQR 12.95–24] arbitrary units [AU]) than the sero-low volunteers (median, 10.54 [IQR, 8.36–12.12] AU) (P = .006). Growth inhibitory activity was significantly higher in the sero-high (median, 21.8% [IQR, 8.15%–29.65%]) than in the sero-low group (median, 8.3% [IQR, 5.6%–10.23%]) (P = .025).ConclusionsCHMI was safe and well tolerated in this population. Individuals with serological evidence of higher malaria exposure were able to better control infection and had higher parasite growth inhibitory activity.Clinical Trials RegistrationNCT03496454.

Background Following successful malaria control during the last decade, Ethiopia instituted a stepwise malaria elimination strategy in selected low-transmission areas. Methods Cross-sectional surveys were conducted in Babile district, Oromia, Ethiopia from July to November 2017 to evaluate malaria infection status using microscopy and nested polymerase chain reaction (nPCR) and serological markers of exposure targeting Plasmodium falciparum and Plasmodium vivax apical membrane antigen-1 (AMA-1). Results Parasite prevalence was 1.2% (14/1135) and 5.1% (58/1143) for P. falciparum and 0.4% (5/1135) and 3.6% (41/1143) for P. vivax by microscopy and nPCR, respectively. Antibody prevalence was associated with current infection by nPCR for both P. falciparum (p

INTRODUCTION: Although there have been several phylogenetic studies on Plasmodium knowlesi (P. knowlesi), only cytochrome c oxidase subunit 1 (COX1) gene analysis has shown some geographical differentiation between the isolates of different countries. METHODS: Phylogenetic analysis of locally acquired P. knowlesi infections, based on circumsporozoite, small subunit ribosomal ribonucleic acid (SSU rRNA), merozoite surface protein 1 and COX1 gene targets, was performed. The results were compared with the published sequences of regional isolates from Malaysia and Thailand. RESULTS: Phylogenetic analysis of the circumsporozoite, SSU rRNA and merozoite surface protein 1 gene sequences for regional P. knowlesi isolates showed no obvious differentiation that could be attributed to their geographical origin. However, COX1 gene analysis showed that it was possible to differentiate between Singapore-acquired P. knowlesi infections and P. knowlesi infections from Peninsular Malaysia and Sarawak, Borneo, Malaysia. CONCLUSION: The ability to differentiate between locally acquired P. knowlesi infections and imported P. knowlesi infections has important utility for the monitoring of P. knowlesi malaria control programmes in Singapore.

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.

Summary Background 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. Methods 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. Findings 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. Interpretation 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. Funding UK Medical Research Council, Natural Environment Research Council, Economic and Social Research Council, and Biotechnology and Biosciences Research Council.

The majority of malaria infections in low transmission settings remain undetectable by conventional diagnostics. A powerful model to identify antibody responses that allow accurate detection of recent exposure to low-density infections is controlled human malaria infection (CHMI) studies in which healthy volunteers are infected with the Plasmodium parasite. We aimed to evaluate antibody responses in malaria-naïve volunteers exposed to a single CHMI using a custom-made protein microarray. All participants developed a blood-stage infection with peak parasite densities up to 100 parasites/μl in the majority of participants (50/54), while the remaining four participants had peak densities between 100 and 200 parasites/μl. There was a strong correlation between parasite density and antibody responses associated with the most reactive blood-stage targets 1 month after CHMI (Etramp 5, GLURP-R2, MSP4 and MSP1-19; Spearman’s ρ = 0.82, p < 0.001). Most volunteers developed antibodies against a potential marker of recent exposure: Etramp 5 (37/45, 82%). Our findings justify validation in endemic populations to define a minimum set of antigens needed to detect exposure to natural low-density infections.

Background: \ud Plasmodium knowlesi is the most common cause of malaria in Malaysian Borneo, with reporting limited to clinical cases presenting to health facilities and scarce data on the true extent of transmission. Serological estimations of transmission have been used with other malaria species to garner information about epidemiological patterns. However, there are a distinct lack of suitable serosurveillance tools for this neglected disease.\ud \ud Methodology/Principal findings: \ud Using in silico tools, we designed and expressed four novel P. knowlesi protein products to address the distinct lack of suitable serosurveillance tools: PkSERA3 antigens 1 and 2, PkSSP2/TRAP and PkTSERA2 antigen 1. Antibody prevalence to these antigens was determined by ELISA for three time-points post-treatment from a hospital-based clinical treatment trial in Sabah, East Malaysia (n = 97 individuals; 241 total samples for all time points). Higher responses were observed for the PkSERA3 antigen 2 (67%, 65/97) across all time-points (day 0: 36.9% 34/92; day 7: 63.8% 46/72; day 28: 58.4% 45/77) with significant differences between the clinical cases and controls (n = 55, mean plus 3 SD) (day 0 p

Background: \ud Primarily impacting poor, rural populations, the zoonotic malaria Plasmodium knowlesi is now the main cause of human malaria within Malaysian Borneo. While data is increasingly available on symptomatic cases, little is known about community-level patterns of exposure and infection. Understanding the true burden of disease and associated risk factors within endemic communities is critical for informing evidence-based control measures.\ud \ud Methodology/Principal findings: \ud We conducted comprehensive surveys in three areas where P. knowlesi transmission is reported: Limbuak, Pulau Banggi and Matunggung, Kudat, Sabah, Malaysia and Bacungan, Palawan, the Philippines. Infection prevalence was low with parasites detected by PCR in only 0.2% (4/2503) of the population. P. knowlesi PkSERA3 ag1 antibody responses were detected in 7.1% (95% CI: 6.2–8.2%) of the population, compared with 16.1% (14.6–17.7%) and 12.6% (11.2–14.1%) for P. falciparum and P. vivax. Sero-prevalence was low in individuals

Prospective studies continue to identify malaria parasite genes with particular patterns of polymorphism which indicate they may be under immune selection, and the encoded proteins require investigation. Sixteen new recombinant protein reagents were designed to characterize three such polymorphic proteins expressed in Plasmodium falciparum schizonts and merozoites: MSPDBL1 (also termed MSP3.4) and MSPDBL2 (MSP3.8), which possess Duffy binding-like (DBL) domains, and SURFIN4.2, encoded by a member of the surface-associated interspersed ( surf ) multigene family. After testing the antigenicities of these reagents by murine immunization and parasite immunofluorescence, we analyzed naturally acquired antibody responses to the antigens in two cohorts in coastal Kenya in which the parasite was endemic (Chonyi [ n = 497] and Ngerenya [ n = 461]). As expected, the prevalence and levels of serum antibodies increased with age. We then investigated correlations with subsequent risk of clinical malaria among children