From contigs towards chromosomes: automatic improvement of long read assemblies (ILRA).

Recent advances in long read technologies not only enable large consortia to aim to sequence all eukaryotes on Earth, but they also allow individual laboratories to sequence their species of interest with relatively low investment. Long read technologies embody the promise of overcoming scaffolding problems associated with repeats and low complexity sequences, but the number of contigs often far exceeds the number of chromosomes and they may contain many insertion and deletion errors around homopolymer tracts. To overcome these issues, we have implemented the ILRA pipeline to correct long read-based assemblies. Contigs are first reordered, renamed, merged, circularized, or filtered if erroneous or contaminated. Illumina short reads are used subsequently to correct homopolymer errors. We successfully tested our approach by improving the genome sequences of Homo sapiens, Trypanosoma brucei, and Leptosphaeria spp., and by generating four novel Plasmodium falciparum assemblies from field samples. We found that correcting homopolymer tracts reduced the number of genes incorrectly annotated as pseudogenes, but an iterative approach seems to be required to correct more sequencing errors. In summary, we describe and benchmark the performance of our new tool, which improved the quality of novel long read assemblies up to 1 Gbp. The pipeline is available at GitHub: https://github.com/ThomasDOtto/ILRA.

Maintenance of high temporal Plasmodium falciparum genetic diversity and complexity of infection in asymptomatic and symptomatic infections in Kilifi, Kenya from 2007 to 2018.

BACKGROUND: High levels of genetic diversity are common characteristics of Plasmodium falciparum parasite populations in high malaria transmission regions. There has been a decline in malaria transmission intensity over 12 years of surveillance in the community in Kilifi, Kenya. This study sought to investigate whether there was a corresponding reduction in P. falciparum genetic diversity, using msp2 as a genetic marker. METHODS: Blood samples were obtained from children (< 15 years) enrolled into a cohort with active weekly surveillance between 2007 and 2018 in Kilifi, Kenya. Asymptomatic infections were defined during the annual cross-sectional blood survey and the first-febrile malaria episode was detected during the weekly follow-up. Parasite DNA was extracted and successfully genotyped using allele-specific nested polymerase chain reactions for msp2 and capillary electrophoresis fragment analysis. RESULTS: Based on cross-sectional surveys conducted in 2007-2018, there was a significant reduction in malaria prevalence (16.2-5.5%: P-value < 0.001), however msp2 genetic diversity remained high. A high heterozygosity index (He) (> 0.95) was observed in both asymptomatic infections and febrile malaria over time. About 281 (68.5%) asymptomatic infections were polyclonal (> 2 variants per infection) compared to 46 (56%) polyclonal first-febrile infections. There was significant difference in complexity of infection (COI) between asymptomatic 2.3 [95% confidence interval (CI) 2.2-2.5] and febrile infections 2.0 (95% CI 1.7-2.3) (P = 0.016). Majority of asymptomatic infections (44.2%) carried mixed alleles (i.e., both FC27 and IC/3D7), while FC27 alleles were more frequent (53.3%) among the first-febrile infections. CONCLUSIONS: Plasmodium falciparum infections in Kilifi are still highly diverse and polyclonal, despite the reduction in malaria transmission in the community.

Antigenic cartography of immune responses to Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1).

Naturally acquired clinical immunity to Plasmodium falciparum is partly mediated by antibodies directed at parasite-derived antigens expressed on the surface of red blood cells which mediate disease and are extremely diverse. Unlike children, adults recognize a broad range of variant surface antigens (VSAs) and are protected from severe disease. Though crucial to the design and feasibility of an effective malaria vaccine, it is not yet known whether immunity arises through cumulative exposure to each of many antigenic types, cross-reactivity between antigenic types, or some other mechanism. In this study, we measured plasma antibody responses of 36 children with symptomatic malaria to a diverse panel of 36 recombinant proteins comprising part of the DBLα domain (the 'DBLα-tag') of PfEMP1, a major class of VSAs. We found that although plasma antibody responses were highly specific to individual antigens, serological profiles of responses across antigens fell into one of just two distinct types. One type was found almost exclusively in children that succumbed to severe disease (19 out of 20) while the other occurred in all children with mild disease (16 out of 16). Moreover, children with severe malaria had serological profiles that were narrower in antigen specificity and shorter-lived than those in children with mild malaria. Borrowing a novel technique used in influenza-antigenic cartography-we mapped these dichotomous serological profiles to amino acid sequence variation within a small sub-region of the PfEMP1 DBLα domain. By applying our methodology on a larger scale, it should be possible to identify epitopes responsible for eliciting the protective version of serological profiles to PfEMP1 thereby accelerating development of a broadly effective anti-disease malaria vaccine.

No evidence of P. falciparum K13 artemisinin conferring mutations over a 24-year analysis in Coastal Kenya, but a near complete reversion to chloroquine wild type parasites.

Antimalarial drug resistance is a substantial impediment to malaria control. The spread of resistance has been described using genetic markers which are important epidemiological tools. We carried out a temporal analysis of changes in allele frequencies of 12 drug resistance markers over two decades of changing antimalarial drug policy in Kenya. We did not detect any of the validated kelch 13 (k13) artemisinin resistance markers, nonetheless, a single k13 allele, K189T, was maintained at a stable high frequency (>10%) over time. There was a distinct shift from chloroquine resistant transporter (crt)-76, multi-drug resistant gene 1 (mdr1)-86 and mdr1-1246 chloroquine (CQ) resistance alleles to a 99% prevalence of CQ sensitive alleles in the population, following the withdrawal of CQ from routine use. In contrast, the dihydropteroate synthetase (dhps) double mutant (437G and 540E) associated with sulfadoxine-pyrimethamine (SP) resistance was maintained at a high frequency (>75%), after a change from SP to artemisinin combination therapies (ACTs). The novel cysteine desulfurase (nfs) K65 allele, implicated in resistance to lumefantrine in a West African study, showed a gradual significant decline in allele frequency pre- and post-ACT introduction (from 38% to 20%), suggesting evidence of directional selection in Kenya, potentially not due to lumefantrine. The high frequency of CQ-sensitive parasites circulating in the population suggests that the re-introduction of CQ in combination therapy for the treatment of malaria can be considered in the future. However, the risk of a re-emergence of CQ resistant parasites circulating below detectable levels or being reintroduced from other regions remains.

Plasmodium falciparum malaria parasite var gene expression is modified by host antibodies: longitudinal evidence from controlled infections of Kenyan adults with varying natural exposure.

BACKGROUND: The PfEMP1 family of Plasmodium falciparum antigens play a key role in pathogenesis of severe malaria through their insertion into the surface of parasite infected erythrocytes, and adhesion to host cells. Previous studies have suggested that parasites expressing PfEMP1 subclasses group A and DC8, associated with severe malaria, may have a growth advantage in immunologically naïve individuals. However, this idea has not been tested in longitudinal studies. METHODS: Here we assessed expression of the var genes encoding PfEMP1, in parasites sampled from volunteers with varying prior exposure to malaria, following experimental infection by sporozoites (PfSPZ). Using qPCR, we tested for associations between the expression of various var subgroups in surviving parasite populations from each volunteer and 1) the levels of participants' antibodies to infected erythrocytes before challenge infection and 2) the apparent in vivo parasite multiplication rate. RESULTS: We show that 1) expression of var genes encoding for group A and DC8-like PfEMP1 were associated with low levels of antibodies to infected erythrocytes (αIE) before challenge, and 2) expression of a DC8-like CIDRα1.1 domain was associated with higher apparent parasite multiplication rate in a manner that was independent of levels of prior antibodies to infected erythrocytes. CONCLUSIONS: This study provides insight into the role of antibodies to infected erythrocytes surface antigens in the development of naturally acquired immunity and may help explain why specific PfEMP1 variants may be associated with severe malaria. TRIAL REGISTRATION: Pan African Clinical Trial Registry: PACTR201211000433272 . Date of registration: 10th October 2012.

Serological Conservation of Parasite-Infected Erythrocytes Predicts Plasmodium falciparum Erythrocyte Membrane Protein 1 Gene Expression but Not Severity of Childhood Malaria.

Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1), expressed on P. falciparum-infected erythrocytes, is a major family of clonally variant targets of naturally acquired immunity to malaria. Previous studies have demonstrated that in areas where malaria is endemic, antibodies to infected erythrocytes from children with severe malaria tend to be more seroprevalent than antibodies to infected erythrocytes from children with nonsevere malaria. These data have led to a working hypothesis that PfEMP1 variants associated with parasite virulence are relatively conserved in structure. However, the longevity of such serologically conserved variants in the parasite population is unknown. Here, using infected erythrocytes from recently sampled clinical P. falciparum samples, we measured serological conservation using pools of antibodies in sera that had been sampled 10 to 12 years earlier. The serological conservation of infected erythrocytes strongly correlated with the expression of specific PfEMP1 subsets previously found to be associated with severe malaria. However, we found no association between serological conservation per se and disease severity within these data. This contrasts with the simple hypothesis that P. falciparum isolates with a serologically conserved group of PfEMP1 variants cause severe malaria. The data are instead consistent with periodic turnover of the immunodominant epitopes of PfEMP1 associated with severe malaria.

The role of PfEMP1 as targets of naturally acquired immunity to childhood malaria: prospects for a vaccine.

The Plasmodium falciparum erythrocyte membrane protein 1 antigens that are inserted onto the surface of P. falciparum infected erythrocytes play a key role both in the pathology of severe malaria and as targets of naturally acquired immunity. They might be considered unlikely vaccine targets because they are extremely diverse. However, several lines of evidence suggest that underneath this molecular diversity there are a restricted set of epitopes which may act as effective targets for a vaccine against severe malaria. Here we review some of the recent developments in this area of research, focusing on work that has assessed the potential of these molecules as possible vaccine targets.

Plasmodium falciparum antigenic variation: relationships between widespread endothelial activation, parasite PfEMP1 expression and severe malaria.

BACKGROUND: Plasmodium falciparum erythrocyte membrane protein 1(PfEMP1) is a family of variant surface antigens (VSA) that mediate the adhesion of parasite infected erythrocytes to capillary endothelial cells within host tissues. Opinion is divided over the role of PfEMP1 in the widespread endothelial activation associated with severe malaria. In a previous study we found evidence for differential associations between defined VSA subsets and specific syndromes of severe malaria: group A-like PfEMP1 expression and the "rosetting" phenotype were associated with impaired consciousness and respiratory distress, respectively. This study explores the involvement of widespread endothelial activation in these associations. METHODS: We used plasma angiopoietin-2 as a marker of widespread endothelial activation. Using logistic regression analysis, we explored the relationships between plasma angiopoietin-2 levels, parasite VSA expression and the two syndromes of severe malaria, impaired consciousness and respiratory distress. RESULTS: Plasma angiopoietin-2 was associated with both syndromes. The rosetting phenotype did not show an independent association with respiratory distress when adjusted for angiopoietin-2, consistent with a single pathogenic mechanism involving widespread endothelial activation. In contrast, group A-like PfEMP1 expression and angiopoietin-2 maintained independent associations with impaired consciousness when adjusted for each other. CONCLUSION: The results are consistent with multiple pathogenic mechanisms leading to severe malaria and heterogeneity in the pathophysiology of impaired consciousness. The observed association between group A-like PfEMP1 and impaired consciousness does not appear to involve widespread endothelial activation.

Linking Cerebral Malaria Pathogenesis to APOE-Mediated Amyloidosis: Observations and Hypothesis.

Although most children with cerebral malaria fully recover, more than a fifth of the survivors develop post-discharge neurodevelopmental sequelae suggestive of advanced neuronal injury. However, the cerebral molecular processes initiating neurological dysfunction in cerebral malaria are still debatable. In this article, we explore available data and hypothesise that cerebral malaria might be linked to APOE-mediated amyloidosis, one of the pathological processes associated with Alzheimer's disease. If our hypothesis is tested and found to be true, it could have far-reaching implications for what we know about cerebral malaria pathogenesis.

The mRNA content of plasma extracellular vesicles provides a window into molecular processes in the brain during cerebral malaria.

The impact of cerebral malaria on the transcriptional profiles of cerebral tissues is difficult to study using noninvasive approaches. We isolated plasma extracellular vesicles (EVs) from patients with cerebral malaria and community controls and sequenced their mRNA content. Deconvolution analysis revealed that EVs from cerebral malaria are enriched in transcripts of brain origin. We ordered the patients with cerebral malaria based on their EV-transcriptional profiles from cross-sectionally collected samples and inferred disease trajectory while using healthy community controls as a starting point. We found that neuronal transcripts in plasma EVs decreased with disease trajectory, whereas transcripts from glial, endothelial, and immune cells increased. Disease trajectory correlated positively with severity indicators like death and was associated with increased VEGFA-VEGFR and glutamatergic signaling, as well as platelet and neutrophil activation. These data suggest that brain tissue responses in cerebral malaria can be studied noninvasively using EVs circulating in peripheral blood.