Dramatic resurgence of malaria after 7 years of intensive vector control interventions in Eastern Uganda.

Background: Tororo District, Uganda experienced a dramatic decrease in malaria burden from 2015-19 following 5 years of indoor residual spraying (IRS) with carbamate (Bendiocarb) and then organophosphate (Actellic) insecticides. However, a marked resurgence occurred in 2020, which coincided with a change to a clothianidin-based IRS formulations (Fludora Fusion/SumiShield). To quantify the magnitude of the resurgence, investigate causes, and evaluate the impact of a shift back to IRS with Actellic in 2023, we assessed changes in malaria metrics in regions within and near Tororo District. Methods: Malaria surveillance data from Nagongera Health Center, Tororo District was included from 2011-2023. In addition, a cohort of 667 residents from 84 houses was followed from August 2020 through September 2023 from an area bordering Tororo and neighboring Busia District, where IRS has never been implemented. Cohort participants underwent passive surveillance for clinical malaria and active surveillance for parasitemia every 28 days. Mosquitoes were collected in cohort households every 2 weeks using CDC light traps. Female Anopheles were speciated and tested for sporozoites and phenotypic insecticide resistance. Temporal comparisons of malaria metrics were stratified by geographic regions. Findings: At Nagongera Health Center average monthly malaria cases varied from 419 prior to implementation of IRS; to 56 after 5 years of IRS with Bendiocarb and Actellic; to 1591 after the change in IRS to Fludora Fusion/SumiShield; to 155 after a change back to Actellic. Among cohort participants living away from the border in Tororo, malaria incidence increased over 8-fold (0.36 vs. 2.97 episodes per person year, p<0.0001) and parasite prevalence increased over 4-fold (17% vs. 70%, p<0.0001) from 2021 to 2022 when Fludora Fusion/SumiShield was used. Incidence decreased almost 5-fold (2.97 vs. 0.70, p<0.0001) and prevalence decreased by 39% (70% vs. 43%, p<0.0001) after shifting back to Actellic. There was a similar pattern among those living near the border in Tororo, with increased incidence between 2021 and 2022 (0.93 vs. 2.40, p<0.0001) followed by a decrease after the change to Actellic (2.40 vs. 1.33, p<0.001). Among residents of Busia, malaria incidence did not change significantly over the 3 years of observation. Malaria resurgence in Tororo was temporally correlated with the replacement of An. gambiae s.s. by An. funestus as the primary vector, with a marked decrease in the density of An. funestus following the shift back to IRS with Actellic. In Busia, An. gambiae s.s. remained the primary vector throughout the observation period. Sporozoite rates were approximately 50% higher among An. funestus compared to the other common malaria vectors. Insecticide resistance phenotyping of An. funestus revealed high tolerance to clothianidin, but full susceptibility to Actellic. Conclusions: A dramatic resurgence of malaria in Tororo was temporally associated with a change to clothianidin-based IRS formulations and emergence of An. funestus as the predominant vector. Malaria decreased after a shift back to IRS with Actellic. This study highlights the ability of malaria vectors to rapidly circumvent control efforts and the importance of high-quality surveillance systems to assess the impact of malaria control interventions and generate timely, actionable data.

Genomic malaria surveillance of antenatal care users detects reduced transmission following elimination interventions in Mozambique.

Routine sampling of pregnant women at first antenatal care (ANC) visits could make Plasmodium falciparum genomic surveillance more cost-efficient and convenient in sub-Saharan Africa. We compare the genetic structure of parasite populations sampled from 289 first ANC users and 93 children from the community in Mozambique between 2015 and 2019. Samples are amplicon sequenced targeting 165 microhaplotypes and 15 drug resistance genes. Metrics of genetic diversity and relatedness, as well as the prevalence of drug resistance markers, are consistent between the two populations. In an area targeted for elimination, intra-host genetic diversity declines in both populations (p = 0.002-0.007), while for the ANC population, population genetic diversity is also lower (p = 0.0004), and genetic relatedness between infections is higher (p = 0.002) than control areas, indicating a recent reduction in the parasite population size. These results highlight the added value of genomic surveillance at ANC clinics to inform about changes in transmission beyond epidemiological data.

Broadly inhibitory antibodies against severe malaria virulence proteins.

Plasmodium falciparum pathology is driven by the accumulation of parasite-infected erythrocytes in microvessels. This process is mediated by the parasite's polymorphic erythrocyte membrane protein 1 (PfEMP1) adhesion proteins. A subset of PfEMP1 variants that bind human endothelial protein C receptor (EPCR) through their CIDRα1 domains is responsible for severe malaria pathogenesis. A longstanding question is whether individual antibodies can recognize the large repertoire of circulating PfEMP1 variants. Here, we describe two broadly reactive and binding-inhibitory human monoclonal antibodies against CIDRα1. The antibodies isolated from two different individuals exhibited a similar and consistent EPCR-binding inhibition of 34 CIDRα1 domains, representing five of the six subclasses of CIDRα1. Both antibodies inhibited EPCR binding of both recombinant full-length and native PfEMP1 proteins as well as parasite sequestration in bioengineered 3D brain microvessels under physiologically relevant flow conditions. Structural analyses of the two antibodies in complex with two different CIDRα1 antigen variants reveal similar binding mechanisms that depend on interactions with three highly conserved amino acid residues of the EPCR-binding site in CIDRα1. These broadly reactive antibodies likely represent a common mechanism of acquired immunity to severe malaria and offer novel insights for the design of a vaccine or treatment targeting severe malaria.

Genetic complexity alters drug susceptibility of asexual and gametocyte stages of Plasmodium falciparum to antimalarial candidates.

Malaria elimination requires interventions able to target both the asexual blood stage (ABS) parasites and transmissible gametocyte stages of Plasmodium falciparum. Lead antimalarial candidates are evaluated against clinical isolates to address key concerns regarding efficacy and to confirm that the current, circulating parasites from endemic regions lack resistance against these candidates. While this has largely been performed on ABS parasites, limited data are available on the transmission-blocking efficacy of compounds with multistage activity. Here, we evaluated the efficacy of lead antimalarial candidates against both ABS parasites and late-stage gametocytes side-by-side, against clinical P. falciparum isolates from southern Africa. We additionally correlated drug efficacy to the genetic diversity of the clinical isolates as determined with a panel of well-characterized, genome-spanning microsatellite markers. Our data indicate varying sensitivities of the isolates to key antimalarial candidates, both for ABS parasites and gametocyte stages. While ABS parasites were efficiently killed, irrespective of genetic complexity, antimalarial candidates lost some gametocytocidal efficacy when the gametocytes originated from genetically complex, multiple-clone infections. This suggests a fitness benefit to multiclone isolates to sustain transmission and reduce drug susceptibility. In conclusion, this is the first study to investigate the efficacy of antimalarial candidates on both ABS parasites and gametocytes from P. falciparum clinical isolates where the influence of parasite genetic complexity is highlighted, ultimately aiding the malaria elimination agenda.

Atypical B cells consist of subsets with distinct functional profiles.

Atypical B cells are a population of activated B cells that are commonly enriched in individuals with chronic immune activation but are also part of a normal immune response to infection or vaccination. To better define the role of atypical B cells in the human adaptive immune response, we performed single-cell sequencing of transcriptomes, cell surface markers, and B cell receptors in individuals with chronic exposure to the malaria parasite Plasmodium falciparum, a condition known to lead to accumulation of circulating atypical B cells. We identified three previously uncharacterized populations of atypical B cells with distinct transcriptional and functional profiles and observed marked differences among these three subsets in their ability to produce immunoglobulin G upon T-cell-dependent activation. Our findings help explain the conflicting observations in prior studies regarding the function of atypical B cells and highlight their different roles in the adaptive immune response in chronic inflammatory conditions.

Genomic malaria surveillance of antenatal care users detects reduced transmission following elimination interventions in Mozambique.

Routine sampling of pregnant women at first antenatal care (ANC) visits could make Plasmodium falciparum genomic surveillance more cost-efficient and convenient in sub-Saharan Africa. We compared the genetic structure of parasite populations sampled from 289 first ANC attendees and 93 children from the community in Mozambique between 2015 and 2019. Samples were amplicon sequenced targeting 165 microhaplotypes and 15 drug resistance genes. Metrics of genetic diversity and relatedness, as well as the prevalence of drug resistance markers, were consistent between the two populations. In an area targeted for elimination, intra-host genetic diversity declined in both populations (p=0.002-0.007), while for the ANC population, population genetic diversity was also lower (p=0.0004), and genetic relatedness between infections were higher (p=0.002) than control areas, indicating a recent reduction in the parasite population size. These results highlight the added value of genomic surveillance at ANC clinics to inform about changes in transmission beyond epidemiological data.

The breadth of the neutralizing antibody response to original SARS-CoV-2 infection is linked to the presence of Long COVID symptoms.

The associations between longitudinal dynamics and the breadth of SARS-CoV-2 neutralizing antibody (nAb) response with various Long COVID phenotypes before vaccination are not known. The capacity of antibodies to cross-neutralize a variety of viral variants may be associated with ongoing pathology and persistent symptoms. We measured longitudinal neutralizing and cross-neutralizing antibody responses to pre- and post-SARS-CoV-2 Omicron variants in participants infected early in the COVID-19 pandemic, before widespread rollout of SARS-CoV-2 vaccines. Cross-sectional regression models adjusted for clinical covariates and longitudinal mixed-effects models were used to determine the impact of the breadth and rate of decay of neutralizing responses on the development of Long COVID symptoms, as well as Long COVID phenotypes. We identified several novel relationships between SARS-CoV-2 antibody neutralization and the presence of Long COVID symptoms. Specifically, we show that, although nAb responses to the original, infecting strain of SARS-CoV-2 were not associated with Long COVID in cross-sectional analyses, cross-neutralization ID50 levels to the Omicron BA.5 variant approximately 4 months following acute infection was independently and significantly associated with greater odds of Long COVID and with persistent gastrointestinal and neurological symptoms. Longitudinal modeling demonstrated significant associations in the overall levels and rates of decay of neutralization capacity with Long COVID phenotypes. A higher proportion of participants had antibodies capable of neutralizing Omicron BA.5 compared with BA.1 or XBB.1.5 variants. Our findings suggest that relationships between various immune responses and Long COVID are likely complex but may involve the breadth of antibody neutralization responses.

Evidence for a role of Anopheles stephensi in the spread of drug- and diagnosis-resistant malaria in Africa.

Anopheles stephensi, an Asian malaria vector, continues to expand across Africa. The vector is now firmly established in urban settings in the Horn of Africa. Its presence in areas where malaria resurged suggested a possible role in causing malaria outbreaks. Here, using a prospective case-control design, we investigated the role of An. stephensi in transmission following a malaria outbreak in Dire Dawa, Ethiopia in April-July 2022. Screening contacts of patients with malaria and febrile controls revealed spatial clustering of Plasmodium falciparum infections around patients with malaria in strong association with the presence of An. stephensi in the household vicinity. Plasmodium sporozoites were detected in these mosquitoes. This outbreak involved clonal propagation of parasites with molecular signatures of artemisinin and diagnostic resistance. To our knowledge, this study provides the strongest evidence so far for a role of An. stephensi in driving an urban malaria outbreak in Africa, highlighting the major public health threat posed by this fast-spreading mosquito.

MOIRE: A software package for the estimation of allele frequencies and effective multiplicity of infection from polyallelic data.

Malaria parasite genetic data can provide insight into parasite phenotypes, evolution, and transmission. However, estimating key parameters such as allele frequencies, multiplicity of infection (MOI), and within-host relatedness from genetic data has been challenging, particularly in the presence of multiple related coinfecting strains. Existing methods often rely on single nucleotide polymorphism (SNP) data and do not account for within-host relatedness. In this study, we introduce a Bayesian approach called MOIRE (Multiplicity Of Infection and allele frequency REcovery), designed to estimate allele frequencies, MOI, and within-host relatedness from genetic data subject to experimental error. Importantly, MOIRE is flexible in accommodating both polyallelic and SNP data, making it adaptable to diverse genotyping panels. We also introduce a novel metric, the effective MOI (eMOI), which integrates MOI and within-host relatedness, providing a robust and interpretable measure of genetic diversity. Using extensive simulations and real-world data from a malaria study in Namibia, we demonstrate the superior performance of MOIRE over naive estimation methods, accurately estimating MOI up to 7 with moderate sized panels of diverse loci (e.g. microhaplotypes). MOIRE also revealed substantial heterogeneity in population mean MOI and mean relatedness across health districts in Namibia, suggesting detectable differences in transmission dynamics. Notably, eMOI emerges as a portable metric of within-host diversity, facilitating meaningful comparisons across settings, even when allele frequencies or genotyping panels are different. MOIRE represents an important addition to the analysis toolkit for malaria population dynamics. Compared to existing software, MOIRE enhances the accuracy of parameter estimation and enables more comprehensive insights into within-host diversity and population structure. Additionally, MOIRE's adaptability to diverse data sources and potential for future improvements make it a valuable asset for research on malaria and other organisms, such as other eukaryotic pathogens. MOIRE is available as an R package at https://eppicenter.github.io/moire/.

An optimized GATK4 pipeline for Plasmodium falciparum whole genome sequencing variant calling and analysis.

BACKGROUND: Accurate variant calls from whole genome sequencing (WGS) of Plasmodium falciparum infections are crucial in malaria population genomics. Here a falciparum variant calling pipeline based on GATK version 4 (GATK4) was optimized and applied to 6626 public Illumina WGS samples. METHODS: Control WGS and accurate PacBio assemblies of 10 laboratory strains were leveraged to optimize parameters that control the heterozygosity, local assembly region size, ploidy, mapping and base quality in both GATK HaplotypeCaller and GenotypeGVCFs. From these controls, a high-quality training dataset was generated to recalibrate the raw variant data. RESULTS: On current high-quality samples (read length = 250 bp, insert size = 405-524 bp), the optimized pipeline shows improved sensitivity (86.6 ± 1.7% for SNPs and 82.2 ± 5.9% for indels) compared to the default GATK4 pipeline (77.7 ± 1.3% for SNPs; and 73.1 ± 5.1% for indels, adjusted P < 0.001) and previous variant calling with GATK version 3 (GATK3, 70.3 ± 3.0% for SNPs and 59.7 ± 5.8% for indels, adjusted P < 0.001). Its sensitivity on simulated mixed infection samples (80.8 ± 6.1% for SNPs and 78.3 ± 5.1% for indels) was again improved relative to default GATK4 (68.8 ± 6.0% for SNPs and 38.9 ± 0.7% for indels, adjusted, adjusted P < 0.001). Precision was high and comparable across all pipelines on each type of data tested. The resulting combination of high-quality SNPs and indels increases the resolution of local population population structure detection in sub-Saharan Africa. Finally, increasing ploidy improves the detection of drug resistance mutations and estimation of complexity of infection. CONCLUSIONS: Overall, this study provides an optimized falciparum GATK4 pipeline resource for variant calling which should help improve genomic studies of malaria.

Autoantigen profiling reveals a shared post-COVID signature in fully recovered and long COVID patients.

Some individuals do not return to baseline health following SARS-CoV-2 infection, leading to a condition known as long COVID. The underlying pathophysiology of long COVID remains unknown. Given that autoantibodies have been found to play a role in severity of SARS-CoV-2 infection and certain other post-COVID sequelae, their potential role in long COVID is important to investigate. Here, we apply a well-established, unbiased, proteome-wide autoantibody detection technology (T7 phage-display assay with immunoprecipitation and next-generation sequencing, PhIP-Seq) to a robustly phenotyped cohort of 121 individuals with long COVID, 64 individuals with prior COVID-19 who reported full recovery, and 57 pre-COVID controls. While a distinct autoreactive signature was detected that separated individuals with prior SARS-CoV-2 infection from those never exposed to SARS-CoV-2, we did not detect patterns of autoreactivity that separated individuals with long COVID from individuals fully recovered from COVID-19. These data suggest that there are robust alterations in autoreactive antibody profiles due to infection; however, no association of autoreactive antibodies and long COVID was apparent by this assay.

Targeted and whole-genome sequencing reveal a north-south divide in P. falciparum drug resistance markers and genetic structure in Mozambique.

Mozambique is one of the four African countries which account for over half of all malaria deaths worldwide, yet little is known about the parasite genetic structure in that country. We performed P. falciparum amplicon and whole genome sequencing on 2251 malaria-infected blood samples collected in 2015 and 2018 in seven provinces of Mozambique to genotype antimalarial resistance markers and interrogate parasite population structure using genome-wide microhaplotyes. Here we show that the only resistance-associated markers observed at frequencies above 5% were pfmdr1-184F (59%), pfdhfr-51I/59 R/108 N (99%) and pfdhps-437G/540E (89%). The frequency of pfdhfr/pfdhps quintuple mutants associated with sulfadoxine-pyrimethamine resistance increased from 80% in 2015 to 89% in 2018 (p < 0.001), with a lower expected heterozygosity and higher relatedness of microhaplotypes surrounding pfdhps mutants than wild-type parasites suggestive of recent selection. pfdhfr/pfdhps quintuple mutants also increased from 72% in the north to 95% in the south (2018; p < 0.001). This resistance gradient was accompanied by a concentration of mutations at pfdhps-436 (17%) in the north, a south-to-north increase in the genetic complexity of P. falciparum infections (p = 0.001) and a microhaplotype signature of regional differentiation. The parasite population structure identified here offers insights to guide antimalarial interventions and epidemiological surveys.

MultiSero: An Open-Source Multiplex-ELISA Platform for Measuring Antibody Responses to Infection.

A multiplexed enzyme-linked immunosorbent assay (ELISA) that simultaneously measures antibody binding to multiple antigens can extend the impact of serosurveillance studies, particularly if the assay approaches the simplicity, robustness, and accuracy of a conventional single-antigen ELISA. Here, we report on the development of multiSero, an open-source multiplex ELISA platform for measuring antibody responses to viral infection. Our assay consists of three parts: (1) an ELISA against an array of proteins in a 96-well format; (2) automated imaging of each well of the ELISA array using an open-source plate reader; and (3) automated measurement of optical densities for each protein within the array using an open-source analysis pipeline. We validated the platform by comparing antibody binding to Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) antigens in 217 human sera samples, showing high sensitivity (0.978), specificity (0.977), positive predictive value (0.978), and negative predictive value (0.977) for classifying seropositivity, a high correlation of multiSero determined antibody titers with commercially available SARS-CoV-2 antibody tests, and antigen-specific changes in antibody titer dynamics upon vaccination. The open-source format and accessibility of our multiSero platform can contribute to the adoption of multiplexed ELISA arrays for serosurveillance studies, for SARS-CoV-2 and other pathogens of significance.

Metagenomic next-generation sequencing to characterize potential etiologies of non-malarial fever in a cohort living in a high malaria burden area of Uganda.

Causes of non-malarial fevers in sub-Saharan Africa remain understudied. We hypothesized that metagenomic next-generation sequencing (mNGS), which allows for broad genomic-level detection of infectious agents in a biological sample, can systematically identify potential causes of non-malarial fevers. The 212 participants in this study were of all ages and were enrolled in a longitudinal malaria cohort in eastern Uganda. Between December 2020 and August 2021, respiratory swabs and plasma samples were collected at 313 study visits where participants presented with fever and were negative for malaria by microscopy. Samples were analyzed using CZ ID, a web-based platform for microbial detection in mNGS data. Overall, viral pathogens were detected at 123 of 313 visits (39%). SARS-CoV-2 was detected at 11 visits, from which full viral genomes were recovered from nine. Other prevalent viruses included Influenza A (14 visits), RSV (12 visits), and three of the four strains of seasonal coronaviruses (6 visits). Notably, 11 influenza cases occurred between May and July 2021, coinciding with when the Delta variant of SARS-CoV-2 was circulating in this population. The primary limitation of this study is that we were unable to estimate the contribution of bacterial microbes to non-malarial fevers, due to the difficulty of distinguishing bacterial microbes that were pathogenic from those that were commensal or contaminants. These results revealed the co-circulation of multiple viral pathogens likely associated with fever in the cohort during this time period. This study illustrates the utility of mNGS in elucidating the multiple potential causes of non-malarial febrile illness. A better understanding of the pathogen landscape in different settings and age groups could aid in informing diagnostics, case management, and public health surveillance systems.

Plasmodium falciparum gametocyte carriage in longitudinally monitored incident infections is associated with duration of infection and human host factors.

Malaria transmission depends on the presence of Plasmodium gametocytes that are the only parasite life stage that can infect mosquitoes. Gametocyte production varies between infections and over the course of infections. Infection duration is highly important for gametocyte production but poorly quantified. Between 2017 and 2019 an all-age cohort of individuals from Tororo, eastern Uganda was followed by continuous passive and routine assessments. We longitudinally monitored 104 incident infections from 98 individuals who were sampled once every 28 days and on any day of symptoms. Among infections that lasted ≥ 3 months, gametocyte appearance was near-universal with 96% of infections having detectable gametocytes prior to clearance. However, most infections were of much shorter duration; 55.7% of asymptomatic infections were detected only once. When considering all asymptomatic infections, regardless of their duration, only 36.3% had detectable gametocytes on at least one time-point prior to parasite clearance. Infections in individuals with sickle-cell trait (HbAS) were more likely to have gametocytes detected (Hazard Rate (HR) = 2.68, 95% CI 1.12, 6.38; p = 0.0231) and had gametocytes detected at higher densities (Density Ratio (DR) = 9.19, 95% CI 2.79, 30.23; p = 0.0002) compared to infections in wildtype (HbAA) individuals. Our findings suggest that a large proportion of incident infections is too short in duration and of too low density to contribute to onward transmission.

Mapping malaria incidence using routine health facility surveillance data in Uganda.

INTRODUCTION: Maps of malaria risk are important tools for allocating resources and tracking progress. Most maps rely on cross-sectional surveys of parasite prevalence, but health facilities represent an underused and powerful data source. We aimed to model and map malaria incidence using health facility data in Uganda. METHODS: Using 24 months (2019-2020) of individual-level outpatient data collected from 74 surveillance health facilities located in 41 districts across Uganda (n=445 648 laboratory-confirmed cases), we estimated monthly malaria incidence for parishes within facility catchment areas (n=310) by estimating care-seeking population denominators. We fit spatio-temporal models to the incidence estimates to predict incidence rates for the rest of Uganda, informed by environmental, sociodemographic and intervention variables. We mapped estimated malaria incidence and its uncertainty at the parish level and compared estimates to other metrics of malaria. To quantify the impact that indoor residual spraying (IRS) may have had, we modelled counterfactual scenarios of malaria incidence in the absence of IRS. RESULTS: Over 4567 parish-months, malaria incidence averaged 705 cases per 1000 person-years. Maps indicated high burden in the north and northeast of Uganda, with lower incidence in the districts receiving IRS. District-level estimates of cases correlated with cases reported by the Ministry of Health (Spearman's r=0.68, p<0.0001), but were considerably higher (40 166 418 cases estimated compared with 27 707 794 cases reported), indicating the potential for underreporting by the routine surveillance system. Modelling of counterfactual scenarios suggest that approximately 6.2 million cases were averted due to IRS across the study period in the 14 districts receiving IRS (estimated population 8 381 223). CONCLUSION: Outpatient information routinely collected by health systems can be a valuable source of data for mapping malaria burden. National Malaria Control Programmes may consider investing in robust surveillance systems within public health facilities as a low-cost, high benefit tool to identify vulnerable regions and track the impact of interventions.

The Breadth of the Neutralizing Antibody Response to Original SARS-CoV-2 Infection is Linked to the Presence of Long COVID Symptoms.

Background: The associations between longitudinal dynamics and the breadth of SARS-CoV-2 neutralizing antibody response with various Long COVID (LC) phenotypes prior to vaccination are not known. The capacity of antibodies to cross neutralize a variety of viral variants may be associated with ongoing pathology and persistent symptoms. Methods: We measured longitudinal neutralizing and cross-neutralizing antibody responses to pre- and post-SARS-CoV-2 Omicron variants in participants infected during the early waves of the COVID-19 pandemic, prior to wide-spread rollout of SARS-CoV-2 vaccines. Cross sectional regression models adjusted for various clinical covariates and longitudinal mixed effects models were used to determine the impact of the breadth and rate of decay of neutralizing responses on the development of Long COVID symptoms in general, as well as LC phenotypes. Results: We identified several novel relationships between SARS-CoV-2 antibody neutralization and the presence of LC symptoms. Specifically, we show that, although neutralizing antibody responses to the original, infecting strain of SARS-CoV-2 were not associated with LC in cross-sectional analyses, cross-neutralization ID50 levels to the Omicron BA.5 variant approximately 4 months following acute infection was independently and significantly associated with greater odds of LC and with persistent gastrointestinal and neurological symptoms. Longitudinal modeling demonstrated significant associations in the overall levels and rates of decay of neutralization capacity with LC phenotypes. A higher proportion of participants had antibodies capable of neutralizing Omicron BA.5 compared with BA.1 or XBB.1.5 variants. Conclusions: Our findings suggest that relationships between various immune responses and LC are likely complex but may involve the breadth of antibody neutralization responses.

SARS-CoV-2 Serology Across Scales: A Framework for Unbiased Estimation of Cumulative Incidence Incorporating Antibody Kinetics and Epidemic Recency.

Serosurveys are a key resource for measuring severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) population exposure. A growing body of evidence suggests that asymptomatic and mild infections (together making up over 95% of all infections) are associated with lower antibody titers than severe infections. Antibody levels also peak a few weeks after infection and decay gradually. We developed a statistical approach to produce estimates of cumulative incidence from raw seroprevalence survey results that account for these sources of spectrum bias. We incorporate data on antibody responses on multiple assays from a postinfection longitudinal cohort, along with epidemic time series to account for the timing of a serosurvey relative to how recently individuals may have been infected. We applied this method to produce estimates of cumulative incidence from 5 large-scale SARS-CoV-2 serosurveys across different settings and study designs. We identified substantial differences between raw seroprevalence and cumulative incidence of over 2-fold in the results of some surveys, and we provide a tool for practitioners to generate cumulative incidence estimates with preset or custom parameter values. While unprecedented efforts have been launched to generate SARS-CoV-2 seroprevalence estimates over this past year, interpretation of results from these studies requires properly accounting for both population-level epidemiologic context and individual-level immune dynamics.

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.

An Optimized GATK4 Pipeline for Plasmodium falciparum Whole Genome Sequencing Variant Calling and Analysis.

Background Accurate variant calls from whole genome sequencing (WGS) of Plasmodium falciparum infections are crucial in malaria population genomics. Here we optimized a falciparum variant calling pipeline based on GATK version 4 (GATK4) and applied it to 6,626 public Illumina WGS samples. Methods We optimized parameters that control the heterozygosity, local assembly region size, ploidy, mapping and base quality in both GATK HaplotypeCaller and GenotypeGVCFs leveraging control WGS and accurate PacBio assemblies of 10 laboratory strains. From these controls we generated a high-quality training dataset to recalibrate the raw variant data. Results On current high-quality samples (read length = 250bp, insert size = 405 - 524 bp ), we show improved sensitivity (86.6 ± 1.7% for SNPs and 82.2 ± 5.9% for indels) compared to the default GATK4 pipeline (77.7 ± 1.3% for SNPs; and 73.1 ± 5.1% for indels, adjusted P < 0.001) and previous variant calling with GATK version 3 (GATK3, 70.3 ± 3.0% for SNPs and 59.7 ± 5.8% for indels, adjusted P < 0.001). The sensitivity of our pipeline on simulated mixed infection samples (80.8 ± 6.1% for SNPs and 78.3 ± 5.1% for indels) was again improved relative to default GATK4 (68.8 ± 6.0% for SNPs and 38.9 ± 0.7% for indels, adjusted P < 0.001). Precision was high and comparable across all pipelines on each type of data tested. We further show that using the combination of high-quality SNPs and indels increases the resolution of local population population structure detection in sub-Saharan Africa. We finally demonstrate that increasing ploidy improves the detection of drug resistance mutations and estimation of complexity of infection. Conclusions Overall, we provide an optimized GATK4 pipeline and resource for falciparum variant calling which should help improve genomic studies of malaria.