Sustained clinical benefit of malaria chemoprevention with sulfadoxine-pyrimethamine (SP) in pregnant women in a region with high SP resistance markers.

OBJECTIVE: The effectiveness of intermittent preventive treatment of malaria in pregnancy with sulfadoxine-pyrimethamine (IPTp-SP) is threatened by increasing SP-resistance in Africa. We assessed the level of SP-resistance markers, and the clinical and parasitological effectiveness of IPTp-SP in southern Mozambique. METHODS: P. falciparum infection, antimalarial antibodies and dhfr/dhps SP-resistance mutants were detected by quantitative polymerase chain reaction (qPCR), suspension array technology and targeted deep sequencing, respectively, among 4016 HIV-negative women in Maputo province (2016-2019). Univariate and multivariate regression models were used to assess the association between taking the recommended three or more IPTp-SP doses (IPTp3+) and parasitological and clinical outcomes. RESULTS: 84.3% (3385/4016) women received three or more IPTp-SP doses. The prevalence of quintuple mutants at first antenatal care (ANC) visit was 94.2%. IPTp3+ was associated with a higher clearance rate of qPCR-detected infections from first ANC visit to delivery (adjusted odds ratio [aOR]=5.9, 95% CI: 1.5-33.3; p = 0.012), lower seroprevalence at delivery of antibodies against the pregnancy-specific antigen VAR2CSADBL34 (aOR=0.72, 95% CI: 0.54-0.95; p = 0.022), and lower prevalence of low birth weight deliveries (aOR: 0.61, 95% CI: 0.41-0.90; p = 0.013). CONCLUSION: A sustained parasitological effect of IPTp-SP contributes to the clinical effectiveness of IPTp3+ in areas with high prevalence of SP-resistance markers.

Expression of the MSPDBL2 antigen in a discrete subset of Plasmodium falciparum schizonts is regulated by GDV1 but may not be linked to sexual commitment.

The Plasmodium falciparum merozoite surface protein MSPDBL2 is a polymorphic antigen targeted by acquired immune responses, and normally expressed in only a minority of mature schizonts. The potential relationship of MSPDBL2 to sexual commitment is examined, as variable mspdbl2 transcript levels and proportions of MSPDBL2-positive mature schizonts in clinical isolates have previously correlated with levels of many sexual stage parasite gene transcripts, although not with the master regulator ap2-g. It is demonstrated that conditional overexpression of the gametocyte development protein GDV1, which promotes sexual commitment, also substantially increases the proportion of MSPDBL2-positive schizonts in culture. Conversely, truncation of the gdv1 gene is shown to prevent any expression of MSPDBL2. However, across diverse P. falciparum cultured lines, the variable proportions of MSPDBL2 positivity in schizonts do not correlate significantly with variable gametocyte conversion rates, indicating it is not involved in sexual commitment. Confirming this, examining a line with endogenous hemagglutinin-tagged AP2-G showed that the individual schizonts expressing MSPDBL2 are mostly different from those expressing AP2-G. Using a selection-linked integration system, modified P. falciparum lines were engineered to express an intact or disrupted version of MSPDBL2, showing the protein is not required for sexual commitment or early gametocyte development. Asexual parasite multiplication rates were also not affected by expression of either intact or disrupted MSPDBL2 in a majority of schizonts. Occurring alongside sexual commitment, the role of the discrete MSPDBL2-positive schizont subpopulation requires further investigation in natural infections where it is under immune selection. IMPORTANCE: Malaria parasites in the blood are remarkably variable, able to switch antigenic targets so they may survive within humans who have already developed specific immune responses. This is one of the challenges in developing vaccines against malaria. MSPDBL2 is a target of naturally acquired immunity expressed in minority proportions of schizonts, the end stages of each 2-day replication cycle in red blood cells which contain merozoites prepared to invade new red blood cells. Results show that the proportion of schizonts expressing MSPDBL2 is positively controlled by the expression of the regulatory gametocyte development protein GDV1. It was previously known that expression of GDV1 leads to increased expression of AP2-G which causes parasites to switch to sexual development, so a surprising finding here is that MSPDBL2-positive parasites are mostly distinct from those that express AP2-G. This discrete antigenic subpopulation of mostly asexual parasites is regulated alongside sexually committed parasites, potentially enabling survival under stress conditions.

Triage practices for emergency care delivery: a qualitative study among febrile patients and healthcare workers in a tertiary care hospital in Nepal.

BACKGROUND: Improving screening and triage practices is essential for early severity assessments at the first point of contact and ensuring timely attention by healthcare workers (HCWs). The main objective of this study was to explore the triage process among febrile patients and HCWs in the emergency department (ED) of a tertiary care hospital in a resource-constrained setting. METHODS: This qualitative study was conducted from March to May 2023 at the ED of Tribhuvan University Teaching Hospital (TUTH), Nepal. The study included in-depth interviews with febrile patients (n = 15) and HCWs (n = 15). Additionally, direct observation notes (n = 20) were collected to document the triage process and patients' experiences in the ED. Data underwent thematic analysis using the Interpretative Phenomenological Analysis (IPA) approach. RESULTS: The ED of TUTH offered comprehensive triage services with clear delineation for the severity of febrile patients in line with the World Health Organization (WHO) guidelines. Nonetheless, challenges and constraints were identified. In the ED, evenings were generally the busiest period, and the triage process was not thorough during night shifts. Perception of triage was limited among patients and variable among HCWs. Digitalizing recordings of patient information including payment was deemed necessary for effective management of patients' waiting times at the triage station. High patient throughput added pressure on HCWs and had a potential influence on the delivery of services. Availability of medical equipment and space were also identified as challenges, with patients sometimes compelled to share beds. There were constraints related to waste disposal, hygiene, cleanliness, and the availability and maintenance of washrooms. Febrile patients experienced delays in receiving timely consultations and laboratory investigation reports, which affected their rapid diagnosis and discharge; nonetheless, patients were satisfied with the overall healthcare services received in the ED. CONCLUSIONS: Improving current triage management requires resource organization, including optimizing the waiting time of patients through a digitalized system. Urgent priorities involve upgrading visitor facilities, patient consultations, laboratory investigations, hygiene, and sanitation. HCWs' recommendations to resource the ED with more equipment, space, and beds and a dedicated triage officer to ensure 24-hour service, together with training and incentives, warrant further attention.

Analytical approaches for antimalarial antibody responses to confirm historical and recent malaria transmission: an example from the Philippines.

Background: Assessing 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. Methods: Here, we evaluated different statistical and machine learning methods for analyzing multiplex malaria-specific antibody response data to classify recent and historical exposure to Plasmodium falciparum and Plasmodium 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 8 P. falciparum and 6 P. vivax-specific antigens from 3 sites with varying transmission intensity. Findings: Measurements of antibody responses and seroprevalence were consistent with the 3 sites' known endemicity status. Among the models tested, a machine learning (ML) approach (Random Forest model) using 4 serological markers (PfGLURP R2, Etramp5.Ag1, GEXP18, and PfMSP119) gave better predictions for P. falciparum recent infection in Palawan (AUC: 0.9591, CI 0.9497-0.9684) than individual antigen seropositivity. Although the ML approach did not improve P. vivax infection predictions, ML classifications confirmed the absence of recent exposure to P. falciparum and P. vivax in both Occidental Mindoro and Bataan. For predicting historical P. falciparum and P. vivax transmission, seroprevalence and seroconversion rates based on cumulative exposure markers AMA1 and MSP119 showed reliable trends in the 3 sites. Interpretation: Our 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. Funding: Newton Fund, Philippine Council for Health Research and Development, UK Medical Research Council.

Serological evaluation of risk factors for exposure to malaria in a pre-elimination setting in Malaysian Borneo.

Malaysia has reported no indigenous cases of P. falciparum and P. vivax for over 3 years. When transmission reaches such low levels, it is important to understand the individuals and locations where exposure risks are high, as they may be at greater risk in the case of a resurgence of transmission. Serology is a useful tool in low transmission settings, providing insight into exposure over longer durations than PCR or RDT. We ran blood samples from a 2015 population-based survey in northern Sabah, Malaysian Borneo on a multiplex bead assay. Using supervised machine learning methods, we characterised recent and historic exposure to Plasmodium falciparum and P. vivax and found recent exposure to P. falciparum to be very low, with exposure to both species increasing with age. We performed a risk-factor assessment on environmental, behavioural, demographic and household factors, and identified forest activity and longer travel times to healthcare as common risk-factors for exposure to P. falciparum and P. vivax. In addition, we used remote-sensing derived data and geostatistical models to assess environmental and spatial associations with exposure. We created predictive maps of exposure to recent P. falciparum in the study area and showed 3 clear foci of exposure. This study provides useful insight into the environmental, spatial and demographic risk factors for P. falciparum and P. vivax at a period of low transmission in Malaysian Borneo. The findings would be valuable in the case of resurgence of human malarias in the region.

Detecting temporal and spatial malaria patterns from first antenatal care visits.

Pregnant women attending first antenatal care (ANC) visits represent a promising malaria surveillance target in Sub-Saharan Africa. We assessed the spatio-temporal relationship between malaria trends at ANC (n = 6471) and in children in the community (n = 3933) and at health facilities (n = 15,467) in southern Mozambique (2016-2019). ANC P. falciparum rates detected by quantitative polymerase chain reaction mirrored rates in children, regardless of gravidity and HIV status (Pearson correlation coefficient [PCC] > 0.8, χ²<1.1), with a 2-3 months lag. Only at rapid diagnostic test detection limits at moderate-to-high transmission, did multigravidae show lower rates than children (PCC = 0.61, 95%CI[-0.12-0.94]). Seroprevalence against the pregnancy-specific antigen VAR2CSA reflected declining malaria trends (PCC = 0.74, 95%CI[0.24-0.77]). 60% (9/15) of hotspots detected from health facility data (n = 6662) using a novel hotspot detector, EpiFRIenDs, were also identified with ANC data (n = 3616). Taken together, we show that ANC-based malaria surveillance offers contemporary information on temporal trends and geographic distribution of malaria burden in the community.

Naturally acquired antibody response to Plasmodium falciparum and Plasmodium vivax among indigenous Orang Asli communities in Peninsular Malaysia.

Malaria remains a public health problem in many parts of the world. In Malaysia, the significant progress towards the national elimination programme and effective disease notification on malaria has resulted in zero indigenous human malaria cases since 2018. However, the country still needs to determine the extent of malaria exposure and transmission patterns, particularly in high-risk populations. In this study, a serological method was used to measure transmission levels of Plasmodium falciparum and Plasmodium vivax among indigenous Orang Asli communities in Kelantan, Peninsular Malaysia. A community-based cross-sectional survey was conducted in three Orang Asli communities (i.e., Pos Bihai, Pos Gob, and Pos Kuala Betis) in Kelantan from June to July 2019. Antibody responses to malaria were assessed by enzyme-linked immunosorbent assay (ELISA) using two P. falciparum (PfAMA-1 and PfMSP-119) and two P. vivax (PvAMA-1 and PvMSP-119) antigens. Age-adjusted antibody responses were analysed using a reversible catalytic model to calculate seroconversion rates (SCRs). Multiple logistic regression was used to investigate factors associated with malaria exposure. The overall malaria seroprevalence was 38.8% for PfAMA-1, 36.4% for PfMSP-119, 2.2% for PvAMA-1, and 9.3% for PvMSP-119. Between study areas, the proportion of seropositivity for any P. falciparum and P. vivax antigens was significantly highest in Pos Kuala Betis with 34.7% (p < 0.001) and 13.6% (p < 0.001), respectively. For all parasite antigens except for PvAMA-1, the proportion of seropositive individuals significantly increased with age (all p < 0.001). Based on the SCR, there was a higher level of P. falciparum transmission than P. vivax in the study area. Multivariate regression analyses showed that living in Pos Kuala Betis was associated with both P. falciparum (adjusted odds ratio [aOR] 5.6, p < 0.001) and P. vivax (aOR 2.1, p < 0.001) seropositivities. Significant associations were also found between age and seropositivity to P. falciparum and P. vivax antigens. Analysis of community-based serological data helps describe the level of transmission, heterogeneity, and factors associated with malaria exposure among indigenous communities in Peninsular Malaysia. This approach could be an important adjunct tool for malaria monitoring and surveillance in low malaria transmission settings in the country.

Prevalence of immunoglobulin G and M to SARS-CoV-2 and other human coronaviruses in The Democratic Republic of Congo, Sierra Leone, and Uganda: A longitudinal study.

OBJECTIVES: We assessed the prevalence of immunoglobulin G (IgG) and IgM against four endemic human coronaviruses and two SARS-CoV-2 antigens among vaccinated and unvaccinated staff at health care centers in Uganda, Sierra Leone, and the Democratic Republic of Congo. METHODS: The government health facility staff who had patient contact in Goma (Democratic Republic of Congo), Kambia District (Sierra Leone), and Masaka District (Uganda) were enrolled. Questionnaires and blood samples were collected at three time points over 4 months. Blood samples were analyzed with the Luminex MAGPIXⓇ. RESULTS: Among unvaccinated participants, the prevalence of IgG/IgM antibodies against SARS-CoV-2 receptor-binding domain or nucleocapsid protein at enrollment was 70% in Goma (138 of 196), 89% in Kambia (112 of 126), and 89% in Masaka (190 of 213). The IgG responses against endemic human coronaviruses at baseline were not associated with SARS-CoV-2 sero-acquisition during follow-up. Among the vaccinated participants, those who had evidence of SARS-CoV-2 IgG/IgM at baseline tended to have higher IgG responses to vaccination than those who were SARS-CoV-2 seronegative at baseline, controlling for the time of sample collection since vaccination. CONCLUSION: The high levels of natural immunity and hybrid immunity should be incorporated into both vaccination policies and prediction models of the impact of subsequent waves of infection in these settings.

Antibodies to repeat-containing antigens in Plasmodium falciparum are exposure-dependent and short-lived in children in natural malaria infections.

Protection against Plasmodium falciparum, which is primarily antibody-mediated, requires recurrent exposure to develop. The study of both naturally acquired limited immunity and vaccine induced protection against malaria remains critical for ongoing eradication efforts. Towards this goal, we deployed a customized P. falciparum PhIP-seq T7 phage display library containing 238,068 tiled 62-amino acid peptides, covering all known coding regions, including antigenic variants, to systematically profile antibody targets in 198 Ugandan children and adults from high and moderate transmission settings. Repeat elements - short amino acid sequences repeated within a protein - were significantly enriched in antibody targets. While breadth of responses to repeat-containing peptides was twofold higher in children living in the high versus moderate exposure setting, no such differences were observed for peptides without repeats, suggesting that antibody responses to repeat-containing regions may be more exposure dependent and/or less durable in children than responses to regions without repeats. Additionally, short motifs associated with seroreactivity were extensively shared among hundreds of antigens, potentially representing cross-reactive epitopes. PfEMP1 shared motifs with the greatest number of other antigens, partly driven by the diversity of PfEMP1 sequences. These data suggest that the large number of repeat elements and potential cross-reactive epitopes found within antigenic regions of P. falciparum could contribute to the inefficient nature of malaria immunity.

Gravidity and malaria trends interact to modify P. falciparum densities and detectability in pregnancy: a 3-year prospective multi-site observational study.

BACKGROUND: Low-density Plasmodium falciparum infections prevail in low transmission settings, where immunity is expected to be minimal, suggesting an immune-independent effect on parasite densities. We aimed to describe parasite densities in pregnancy, and determine how gravidity and antibody-mediated immunity affect these, during a period of declining malaria transmission in southern Mozambique. METHODS: We documented P. falciparum infections at first antenatal care visits (n = 6471) between November 2016 and October 2019 in Ilha Josina (high-to-moderate transmission area), Manhiça (low transmission area), and Magude (pre-elimination area). Two-way interactions in mixed-effects regression models were used to assess gravidity-dependent differences in quantitative PCR-determined P. falciparum positivity rates (PfPRqPCR) and densities, in the relative proportion of detectable infections (pDi) with current diagnostic tests (≥ 100 parasites/µL) and in antimalarial antibodies. RESULTS: PfPRqPCR declined from 28 to 13% in Ilha Josina and from 5-7 to 2% in Magude and Manhiça. In primigravidae, pDi was highest in Ilha Josina at the first study year (p = 0.048), which declined with falling PfPRqPCR (relative change/year: 0.41, 95% CI [0.08; 0.73], p = 0.029), with no differences in antibody levels. Higher parasite densities in primigravidae from Ilha Josina during the first year were accompanied by a larger reduction of maternal hemoglobin levels (- 1.60, 95% CI [- 2.49; - 0.72; p < 0.001), than in Magude (- 0.76, 95% CI [- 1.51; - 0.01]; p = 0.047) and Manhiça (- 0.44, 95% CI [- 0.99; 0.10; p = 0.112). In contrast, multigravidae during the transmission peak in Ilha Josina carried the lowest pDi (p = 0.049). As PfPRqPCR declined, geometric mean of parasite densities increased (4.63, 95% CI [1.28; 16.82], p = 0.020), and antibody levels declined among secundigravidae from Ilha Josina. CONCLUSIONS: The proportion of detectable and clinically relevant infections is the highest in primigravid women from high-to-moderate transmission settings and decreases with declining malaria. In contrast, the falling malaria trends are accompanied by increased parasite densities and reduced humoral immunity among secundigravidae. Factors other than acquired immunity thus emerge as potentially important for producing less detectable infections among primigravidae during marked declines in malaria transmission.

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.

Seroepidemiological surveillance, community perceptions and associated risk factors of malaria exposure among forest-goers in Northeastern Thailand.

Malaria remains a major public health challenge in Thailand. Continuous assessment and understanding of the behavior and perceptions related to malaria exposure in the high-risk group are necessary to achieve the elimination goal. This study aimed to investigate the parasite prevalence, seroprevalence rate, knowledge, attitudes, and practices (KAP), and malaria risk factors in rural communities living close to a forested area in the northeastern part of Thailand. A community-based cross-sectional survey was conducted in three forest-goer communities (i.e., Ban Khok, Ban Koh, and Dong Yang) located in Khamcha-i district, Mukdahan Province, Thailand, from July to August 2019. Demographic, socioeconomic information and KAP data were collected using a structured questionnaire. Parasite prevalence was determined by microscopy. Seroprevalence was determined via ELISA using two Plasmodium falciparum (PfAMA-1 and PfMSP-119) and two Plasmodium vivax (PvAMA-1 and PvMSP-119) antigens. Age-adjusted antibody responses were analyzed using a reversible catalytic model to calculate seroconversion rate (SCR). Malaria parasite was not detected in any of the 345 participants. The overall malaria seroprevalence was 72.2% for PfAMA-1, 18.8% for PfMSP-119, 32.5% for PvAMA-1, and 4.4% for PvMSP-119. The proportion of seroprevalence for P. falciparum and P. vivax antigens was significantly highest in Ban Koh (35.1%, P < 0.001) and Don Yang (18.8%, P < 0.001), respectively. For all parasite antigens except PvMSP-119, the proportion of seropositive individuals significantly increased with age (P < 0.001). Based on the SCRs, there was a higher level of P. falciparum transmission than P. vivax. Regarding KAP, almost all respondents showed adequate knowledge and awareness about malaria. Nevertheless, significant effort is needed to improve positive attitudes and practices concerning malaria prevention measures. Multivariate regression analyses showed that living in Ban Koh was associated with both P. falciparum (adjusted odds ratio [aOR] 12.87, P < 0.001) and P. vivax (aOR 9.78, P < 0.001) seropositivities. We also found significant associations between age and seropositivity against P. falciparum and P. vivax antigens. The data suggest that seroepidemiological surveillance using AMA-1 and MSP-119 antigens may provide further evidence to reconstruct malaria exposure history. The absence of weak evidence of recent malaria transmission in Mukdahan Province is promising in the context of the disease elimination program.

Highly Variable Expression of Merozoite Surface Protein MSPDBL2 in Diverse Plasmodium falciparum Clinical Isolates and Transcriptome Scans for Correlating Genes.

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 analyzed in the first ex vivo culture cycle of 96 clinical isolates from 4 populations with various 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, but the frequency distribution was highly skewed, as nine isolates had more than 3% schizonts positive and one had 73% positive. To investigate whether the expression of other gene loci correlated with MSPDBL2 expression, whole-transcriptome sequencing was performed on schizont-enriched material from 17 of the isolates with a wide range of proportions of schizonts positive. Transcripts of particular genes were highly significantly positively correlated with MSPDBL2 positivity in schizonts as well as with mspdbl2 gene transcript levels, showing overrepresentation of genes implicated previously as involved in gametocytogenesis but not including the gametocytogenesis master regulator ap2-g. Single-cell transcriptome analysis of a laboratory-adapted clone showed that most individual parasites expressing mspdbl2 did not express ap2-g, consistent with MSPDBL2 marking a developmental subpopulation that is distinct but likely to co-occur alongside sexual commitment. IMPORTANCE These findings contribute to understanding malaria parasite antigenic and developmental variation, focusing on the merozoite surface protein encoded by the single locus under strongest balancing selection. Analyzing the initial ex vivo generation of parasites grown from a wide sample of clinical infections, we show a unique and highly skewed pattern of natural expression frequencies of MSPDBL2, distinct from that of any other antigen. Bulk transcriptome analysis of a range of clinical isolates showed significant overrepresentation of sexual development genes among those positively correlated with MSPDBL2 protein and mspdbl2 gene expression, indicating the MSPDBL2-positive subpopulation to be often coincident with parasites developing sexually in preparation for transmission. Single-cell transcriptome data confirm the absence of a direct correlation with the ap2-g master regulator of sexual development, indicating that the MSPDBL2-positive subpopulation has a separate function in asexual survival and replication under conditions that promote terminal sexual differentiation.

Plasmodium falciparum serology: A comparison of two protein production methods for analysis of antibody responses by protein microarray.

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 clear correlation 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.

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.

Sero-epidemiological study of arbovirus infection following the 2015-2016 Zika virus outbreak in Cabo Verde.

In November 2015, cases of Zika virus infection were recorded in Cabo Verde (Africa), originating from Brazil. The outbreak subsided after seven months with 7580 suspected cases. We performed a serological survey (n = 431) in Praia, the capital city, 3 months after transmission ceased. Serum samples were screened for arbovirus antibodies using ELISA techniques and revealed seroconverted individuals with Zika (10.9%), dengue (1-4) (12.5%), yellow fever (0.2%) and chikungunya (2.6%) infections. Zika seropositivity was predominantly observed amongst females (70%). Using a logistic model, risk factors for increased odds of Zika seropositivity included age, self-reported Zika infection, and dengue seropositivity. Serological data from Zika and dengue virus assays were strongly correlated (Spearman's rs = 0.80), which reduced when using a double antigen binding ELISA (Spearman's rs = 0.54). Overall, our work improves an understanding of how Zika and other arboviruses have spread throughout the Cabo Verde population. It also demonstrates the utility of serological assay formats for outbreak investigations.

Serological evaluation of the effectiveness of reactive focal mass drug administration and reactive vector control to reduce malaria transmission in Zambezi Region, Namibia: Results from a secondary analysis of a cluster randomised trial.

BACKGROUND: Due to challenges in measuring changes in malaria at low transmission, serology is increasingly being used to complement clinical and parasitological surveillance. Longitudinal studies have shown that serological markers, such as Etramp5.Ag1, can reflect spatio-temporal differences in malaria transmission. However, these markers have yet to be used as endpoints in intervention trials. METHODS: Based on data from 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), this study conducted a secondary analysis comparing antibody responses between intervention arms as trial endpoints. Antibody responses were measured on a multiplex immunoassay, using a panel of eight serological markers of Plasmodium falciparum infection - Etramp5.Ag1, GEXP18, HSP40.Ag1, Rh2.2030, EBA175, PfMSP119, PfAMA1, and PfGLURP.R2. FINDINGS: 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 prevalence ratio (aPR) for Etramp5.Ag1 was 0.78 (95%CI 0.65 - 0.91, p = 0.0007) in the rfMDA arms and 0.79 (95%CI 0.67 - 0.92, p = 0.001) in the RAVC arms. For the combined rfMDA plus RAVC intervention, aPR was 0.59 (95%CI 0.46 - 0.76, p < 0.0001). Significant reductions were also observed based on continuous antibody responses. Sero-prevalence as an endpoint was found to achieve higher study power (99.9% power to detect a 50% reduction in prevalence) compared to quantitative polymerase chain reaction (qPCR) prevalence (72.9% power to detect a 50% reduction in prevalence). INTERPRETATION: While the observed relative reduction in qPCR prevalence in the study was greater than serology, the use of serological endpoints to evaluate trial outcomes measured effect size with improved precision and study power. 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. FUNDING: This study was supported by Novartis Foundation (A122666), the Bill & Melinda Gates Foundation (OPP1160129), and the Horchow Family Fund (5,300,375,400).

Reactive focal drug administration associated with decreased malaria transmission in an elimination setting: Serological evidence from the cluster-randomized CoRE study.

Efforts to eliminate malaria transmission need evidence-based strategies. However, accurately assessing end-game malaria elimination strategies is challenging due to the low level of transmission and the rarity of infections. We hypothesised that presumptively treating individuals during reactive case detection (RCD) would reduce transmission and that serology would more sensitively detect this change over standard approaches. We conducted a cluster randomised control trial (NCT02654912) of presumptive reactive focal drug administration (RFDA-intervention) compared to the standard of care, reactive focal test and treat (RFTAT-control) in Southern Province, Zambia-an area of low seasonal transmission (overall incidence of ~3 per 1,000). We measured routine malaria incidence from health facilities as well as PCR parasite prevalence / antimalarial seroprevalence in an endline cross-sectional population survey. No significant difference was identified from routine incidence data and endline prevalence by polymerase chain reaction (PCR) had insufficient numbers of malaria infections (i.e., 16 infections among 6,276 children) to assess the intervention. Comparing long-term serological markers, we found a 19% (95% CI = 4-32%) reduction in seropositivity for the RFDA intervention using a difference in differences approach incorporating serological positivity and age. We also found a 37% (95% CI = 2-59%) reduction in seropositivity to short-term serological markers in a post-only comparison. These serological analyses provide compelling evidence that RFDA both has an impact on malaria transmission and is an appropriate end-game malaria elimination strategy. Furthermore, serology provides a more sensitive approach to measure changes in transmission that other approaches miss, particularly in very low transmission settings. Trial Registration: Registered at www.clinicaltrials.gov (NCT02654912, 13/1/2016).

Plasmodium knowlesi detection methods for human infections-Diagnosis and surveillance.

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.

Rapid Screening for Non-falciparum Malaria in Elimination Settings Using Multiplex Antigen and Antibody Detection: Post Hoc Identification of Plasmodium malariae in an Infant in Haiti.

Haiti is targeting malaria elimination by 2025. The Grand'Anse department in southwestern Haiti experiences one-third to half of all nationally reported Plasmodium falciparum cases. Although there are historical reports of Plasmodium vivax and Plasmodium malariae, today, non-falciparum infections would remain undetected because of extensive use of falciparum-specific histidine-rich protein 2 (HRP2) rapid diagnostic tests (RDT) at health facilities. A recent case-control study was conducted in Grand'Anse to identify risk factors for P. falciparum infection using HRP2-based RDTs (n = 1,107). Post hoc multiplex Plasmodium antigenemia and antibody (IgG) detection by multiplex bead assay revealed one blood sample positive for pan-Plasmodium aldolase, negative for P. falciparum HRP2, and positive for IgG antibodies to P. malariae. Based on this finding, we selected 52 samples with possible P. malariae infection using IgG and antigenemia data and confirmed infection status by species-specific PCR. We confirmed one P. malariae infection in a 6-month-old infant without travel history. Congenital P. malariae could not be excluded. However, our finding-in combination with historical reports of P. malariae-warrants further investigation into the presence and possible extent of non-falciparum malaria in Haiti. Furthermore, we showed the use of multiplex Plasmodium antigen and IgG detection in selecting samples of interest for subsequent PCR analysis, thereby reducing costs as opposed to testing all available samples by PCR. This is of specific use in low-transmission or eliminating settings where infections are rare.