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OBJECTIVES/GOALS: Recent in vitro evidence suggests that diverse parasite protein families called RIFINs and STEVORs are displayed on the surface of infected red blood cells and may have a role in severe malaria, but they remain sparsely studied in natural infections. We measured the RNA expression of these antigens in Malian children with severe or mild malaria illness. METHODS/STUDY POPULATION: We collected blood samples from Malian children aged six months to five years, including 14 with cerebral malaria, 10 with severe malarial anemia, and demographic-matched controls with mild, uncomplicated malaria. We extracted total RNA from each patient and used a custom capture array to selectively enrich Plasmodium falciparum parasite RNA. We then performed Illumina next-generation RNA sequencing and reconstructed parasite transcriptomes using reference-free de novo assembly. We identified RNA encoding RIFINs and STEVORs using an in-house classifier, then measured the diversity and abundance of gene expression for each infection. Expression diversity was defined as the number of unique variants transcribed. Expression abundance was calculated as transcripts per million (TPM). RESULTS/ANTICIPATED RESULTS: Cerebral malaria cases, but not severe malarial anemia cases, had higher diversity and abundance of RIFIN expression compared to mild infections. Type A RIFINs predominated over Type B RIFINs, and the same two RIFINs were predominantly expressed in all disease phenotypes. We anticipate that predominantly expressed RIFINs share high sequence homology with variants previously shown to bind blood antigens or immune inhibitory receptors. STEVOR expression was also higher in cerebral malaria compared to mild malaria, but STEVOR transcripts were sparse overall. DISCUSSION/SIGNIFICANCE: Elevated RIFIN expression in cerebral malaria over mild malaria supports a role for these antigens in pathogenesis. Severe malarial anemia may progress through a different pathogenic mechanism. Predominantly expressed RIFIN variants may be promising targets for vaccines and therapeutics to protect children against cerebral malaria.

The discovery and development of transmission-blocking therapies challenge malaria elimination and necessitate standard and reproducible bioassays to measure the blocking properties of antimalarial drugs and candidate compounds. Most of the current bioassays evaluating the transmission-blocking activity of compounds rely on laboratory-adapted Plasmodium strains.

The discovery and development of transmission-blocking therapies challenge malaria elimination and necessitate standard and reproducible bioassays to measure the blocking properties of antimalarial drugs and candidate compounds. Most of the current bioassays evaluating the transmission-blocking activity of compounds rely on laboratory-adapted

Plasmodium falciparum malaria cases in Africa represent over 90% of the global burden with Mali being amongst the 11 highest burden countries that account for 70% of this annual incidence. The persistence of P. falciparum despite massive global interventions is because of its genetic diversity that drives its ability to adapt to environmental changes, develop resistance to drugs, and evade the host immune system. Knowledge on P. falciparum genetic diversity across populations and intervention landscape is thus critical for the implementation of new strategies to eliminate malaria. This study assessed genetic variation with 12,177 high-quality SNPs from 830 Malian P. falciparum isolates collected between 2007 and 2017 from seven locations. The complexity of infections remained high, varied between sites, and showed a trend toward overall decreasing complexity over the decade. Though there was no significant substructure, allele frequencies varied geographically, partly driven by temporal variance in sampling, particularly for drug resistance and antigen loci. Thirty-two mutations in known drug resistance markers (pfcrt, pfdhps, pfdhfr, pfmdr1, pfmdr2, and pfk13) attained a frequency of at least 2% in the populations. SNPs within and around the major markers of resistance to quinolines (pfmdr1 and pfcrt) and antifolates (pfdhfr and pfdhps) varied temporally and geographically, with strong linkage disequilibrium and signatures of directional selection in the genome. These geo-temporal populations also differentiated at alleles in immune-related loci, including, protein E140, pfsurfin8, pfclag8, and pfceltos, as well as pftrap, which showed signatures of haplotype differentiation between populations. Several regions across the genomes, including five known drug resistance loci, showed signatures of differential positive selection. These results suggest that drugs and immune pressure are dominant selective forces against P. falciparum in Mali, but their effect on the parasite genome varies temporally and spatially. Interventions interacting with these genomic variants need to be routinely evaluated as malaria elimination strategies are implemented.

Structural biology is an essential tool for understanding the molecular basis of diseases, which can guide the rational design of new drugs, vaccines, and the optimisation of existing medicines. However, most African countries do not conduct structural biology research due to limited resources, lack of trained persons, and an exodus of skilled scientists. The most urgent requirement is to build on the emerging centres in Africa – some well-established, others growing. This can be achieved through workshops that improve networking, grow skills, and develop mechanisms for access to light source beamlines for defining X-ray structures across the continent. These would encourage the growth of structural biology, which is central to understanding biological functions and developing new antimicrobials and other drugs. In this light, a hands-on training workshop in structural biology series 4 was organised by BioStruct-Africa and the Malaria Research and Training Center (MRTC) in Bamako, Mali, to help bridge this gap. The workshop was hosted by MRTC from the 25th to 28th of April 2022. Through a series of lectures and practicals, the workshop enlightened the participants on how structural biology can be utilised to find solutions to the prevalent diseases in Africa. The short training gave them an overview of target selection, protein production and purification, structural determination techniques, and analysis in combination with high-throughput, structure-guided, fragment-based drug design.

Controlled human malaria infections (CHMI) are a valuable tool to study parasite gene expressionin vivounder defined conditions. In previous studies, virulence gene expression was analyzed in samples from volunteers infected with thePlasmodium falciparum(Pf) NF54 isolate, which is of African origin. Here, we provide an in-depth investigation of parasite virulence gene expression in malaria-naïve European volunteers undergoing CHMI with the genetically distinct Pf 7G8 clone, originating in Brazil. Differential expression ofvargenes, encoding major virulence factors of Pf, PfEMP1s, was assessed inex vivoparasite samples as well as in parasites from thein vitrocell bank culture that was used to generate the sporozoites (SPZ) for CHMI (Sanaria® PfSPZ Challenge (7G8)). We report broad activation of mainly B-type subtelomeric locatedvargenes at the onset of a 7G8 blood stage infection in naïve volunteers, mirroring the NF54 expression study and suggesting that the expression of virulence-associated genes is generally reset during transmission from the mosquito to the human host. However, in 7G8 parasites, we additionally detected a continuously expressed single C-type variant, Pf7G8_040025600, that was most highly expressed in both pre-mosquito cell bank and volunteer samples, suggesting that 7G8, unlike NF54, maintains expression of some previously expressedvarvariants during transmission. This suggests that in a new host, the parasite may preferentially express the variants that previously allowed successful infection and transmission.

var genes encode Plasmodium falciparum erythrocyte membrane protein-1 (PfEMP1) antigens. These highly diverse antigens are displayed on the surface of infected erythrocytes and play a critical role in immune evasion and sequestration of infected erythrocytes. Studies of var expression using non-leukocyte-depleted blood are challenging because of the predominance of host genetic material and lack of conserved var segments. Our goal was to enrich for parasite RNA, allowing de novo assembly of var genes and detection of expressed novel variants. We used two overall approaches: (i) enriching for total mRNA in the sequencing library preparations and (ii) enriching for parasite RNA with a custom capture array based on Roche’s SeqCap EZ enrichment system. The capture array was designed with probes based on the whole 3D7 reference genome and an additional >4,000 full-length var gene sequences from other P. falciparum strains. We tested each method on RNA samples from Malian children with severe or uncomplicated malaria infections. All reads mapping to the human genome were removed, the remaining reads were assembled de novo into transcripts, and from these, var-like transcripts were identified and annotated. The capture array produced the longest maximum length and largest numbers of var gene transcripts in each sample, particularly in samples with low parasitemia. Identifying the most-expressed var gene sequences in whole-blood clinical samples without the need for extensive processing or generating sample-specific reference genome data is critical for understanding the role of PfEMP1s in malaria pathogenesis. IMPORTANCE Malaria parasites display antigens on the surface of infected red blood cells in the human host that facilitate attachment to blood vessels, contributing to the severity of infection. These antigens are highly variable, allowing the parasite to evade the immune system. Identifying these expressed antigens is critical to understanding the development of severe malarial disease. However, clinical samples contain limited amounts of parasite genetic material, a challenge for sequencing efforts further compounded by the extreme diversity of the parasite surface antigens. We present a method that enriches for these antigen sequences in clinical samples using a custom capture array, requiring minimal processing in the field. While our results are focused on the malaria parasite Plasmodium falciparum, this approach has broad applicability to other highly diverse antigens from other parasites and pathogens such as those that cause giardiasis and leishmaniasis.

Background Drug resistance is one of the greatest challenges of malaria control programme in Mali. Recent advances in next-generation sequencing (NGS) technologies provide new and effective ways of tracking drug-resistant malaria parasites in Africa. The diversity and the prevalence of Plasmodium falciparum drug-resistance molecular markers were assessed in Dangassa and Nioro-du-Sahel in Mali, two sites with distinct malaria transmission patterns. Dangassa has an intense seasonal malaria transmission, whereas Nioro-du-Sahel has an unstable and short seasonal malaria transmission. Methods Up to 270 dried blood spot samples (214 in Dangassa and 56 in Nioro-du-Sahel) were collected from P. falciparum positive patients in 2016. Samples were analysed on the Agena MassARRAY® iPLEX platform. Specific codons were targeted in Pfcrt, Pfmdr1, Pfdhfr, and Pfdhps, Pfarps10, Pfferredoxin, Pfexonuclease and Pfmdr2 genes. The Sanger’s 101-SNPs-barcode method was used to assess the genetic diversity of P. falciparum and to determine the parasite species. Results The Pfcrt_76T chloroquine-resistance genotype was found at a rate of 64.4% in Dangassa and 45.2% in Nioro-du-Sahel (p = 0.025). The Pfdhfr_51I-59R-108N pyrimethamine-resistance genotype was 14.1% and 19.6%, respectively in Dangassa and Nioro-du-Sahel. Mutations in the Pfdhps_S436-A437-K540-A581-613A sulfadoxine-resistance gene was significantly more prevalent in Dangassa as compared to Nioro-du-Sahel (p = 0.035). Up to 17.8% of the isolates from Dangassa vs 7% from Nioro-du-Sahel harboured at least two codon substitutions in this haplotype. The amodiaquine-resistance Pfmdr1_N86Y mutation was identified in only three samples (two in Dangassa and one in Nioro-du-Sahel). The lumefantrine-reduced susceptibility Pfmdr1_Y184F mutation was found in 39.9% and 48.2% of samples in Dangassa and Nioro-du-Sahel, respectively. One piperaquine-resistance Exo_E415G mutation was found in Dangassa, while no artemisinin resistance genetic-background were identified. A high P. falciparum diversity was observed, but no clear genetic aggregation was found at either study sites. Higher multiplicity of infection was observed in Dangassa with both COIL (p = 0.04) and Real McCOIL (p = 0.02) methods relative to Nioro-du-Sahel. Conclusions This study reveals high prevalence of chloroquine and pyrimethamine-resistance markers as well as high codon substitution rate in the sulfadoxine-resistance gene. High genetic diversity of P. falciparum was observed. These observations suggest that the use of artemisinins is relevant in both Dangassa and Nioro-du-Sahel.

Background Plasmodium falciparum erythrocyte membrane protein-1 (PfEMP1) antigens play a critical role in host immune evasion. Serologic responses to these antigens have been associated with protection from clinical malaria, suggesting that antibodies to PfEMP1 antigens may contribute to natural immunity. The first N-terminal constitutive domain in a PfEMP1 is the Duffy binding-like alpha (DBL-α) domain, which contains a 300 to 400 base pair region unique to each particular protein (the DBL-α “tag”). This DBL-α tag has been used as a marker of PfEMP1 diversity and serologic responses in malaria-exposed populations. In this study, using sera from a malaria-endemic region, responses to DBL-α tags were compared to responses to the corresponding entire DBL-α domain (or “parent” domain) coupled with the succeeding cysteine-rich interdomain region (CIDR). Methods A protein microarray populated with DBL-α tags, the parent DBL-CIDR head structures, and downstream PfEMP1 protein fragments was probed with sera from Malian children (aged 1 to 6 years) and adults from the control arms of apical membrane antigen 1 (AMA1) vaccine clinical trials before and during a malaria transmission season. Serological responses to the DBL-α tag and the DBL-CIDR head structure were measured and compared in children and adults, and throughout the season. Results Malian serologic responses to a PfEMP1’s DBL-α tag region did not correlate with seasonal malaria exposure, or with responses to the parent DBL-CIDR head structure in either children or adults. Parent DBL-CIDR head structures were better indicators of malaria exposure. Conclusions Larger PfEMP1 domains may be better indicators of malaria exposure than short, variable PfEMP1 fragments such as DBL-α tags. PfEMP1 head structures that include conserved sequences appear particularly well suited for study as serologic predictors of malaria exposure. Electronic supplementary material The online version of this article (10.1186/s12936-019-2905-9) contains supplementary material, which is available to authorized users.

Background Artemisinin-based combination therapy (ACT) was deployed in 2005 as an alternative to chloroquine and is considered the most efficacious treatment currently available for uncomplicated falciparum malaria. While widespread artemisinin resistance has not been reported to date in Africa, recent studies have reported partial resistance in Rwanda. The purpose of this study is to provide a current systematic review and meta-analysis on ACT at Mali study sites, where falciparum malaria is highly endemic. Methods A systematic review of the literature maintained in the bibliographic databases accessible through the PubMed, ScienceDirect and Web of Science search engines was performed to identify research studies on ACT occurring at Mali study sites. Selected studies included trials occurring at Mali study sites with reported polymerase chain reaction (PCR)-corrected adequate clinical and parasite response rates (ACPRcs) at 28 days. Data were stratified by treatment arm (artemether–lumefantrine (AL), the first-line treatment for falciparum malaria in Mali and non-AL arms) and analysed using random-effects, meta-analysis approaches. Results A total of 11 studies met the inclusion criteria, and a risk of bias assessment carried out by two independent reviewers determined low risk of bias among all assessed criteria. The ACPRc for the first-line AL at Mali sites was 99.0% (95% CI (98.3%, 99.8%)), while the ACPRc among non-AL treatment arms was 98.9% (95% CI (98.3%, 99.5%)). The difference in ACPRcs between non-AL treatment arms and AL treatment arms was not statistically significant (p = .752), suggesting that there are potential treatment alternatives beyond the first-line of AL in Mali. Conclusions ACT remains highly efficacious in treating uncomplicated falciparum malaria in Mali. Country-specific meta-analyses on ACT are needed on an ongoing basis for monitoring and evaluating drug efficacy patterns to guide local malaria treatment policies, particularly in the wake of observed artemisinin resistance in Southeast Asia and partial resistance in Rwanda.

Next generation sequencing (NGS) has become a necessary tool for genomic epidemiology. Even though the utility of genomics in human health has been proved, the genomic surveillance has never been so important until the COVID 19 pandemic. This has been evidenced with the detection of new variants of SARS-CoV-2 in the United Kingdom, South Africa and Brazil recently using genomic surveillance. Until recently, Malian scientists did not have access to any local NGS platform and samples had to be shipped abroad for sequencing. Here, we report on how we adapted a laboratory setup for Plasmodium research to generate the first complete SARS-CoV-2 genome locally. Total RNA underwent a library preparation using an Illumina TruSeq stranded RNA kit. A metagenomics sequencing was performed on an Illumina MiSeq platform following by bioinformatic analyses on a local server in Mali. We recovered a full genome of SARS-CoV-2 of 29 kb with an average depth coverage of 200x. We have demonstrated our capability of generating a high quality genome with limited resources and highlight the need to develop genomics capacity locally to solve health problems. We discuss challenges related to access to reagents during a pandemic period and propose some home-made solutions.

Background Artemether–lumefantrine (AL) and artesunate–amodiaquine are first-line treatment for uncomplicated malaria in many endemic countries, including Mali. Dihydroartemisinin–piperaquine (DHA–PQ) is also an alternative first-line artemisinin-based combination therapy, but only few data are available on DHA–PQ efficacy in sub-Saharan Africa. The main aim of this study was to compare clinical efficacy of DHA–PQ versus AL, using the World Health Organization (WHO) 42-day in vivo protocol. Methods The efficacy of three-dose regimens of DHA–PQ was compared to AL combination in a randomized, comparative open label trial using the WHO 42-day follow-up protocol from 2013 to 2015 in Doneguebougou and Torodo, Mali. The primary endpoint was to access the PCR-corrected Adequate Clinical and Parasitological Responses at day 28. Results A total of 317 uncomplicated malaria patients were enrolled, with 159 in DHA–PQ arm and 158 in AL arm. The parasite positivity rate decreased from 68.4% (95% CI 60.5–75.5) on day 1 to 3.8% (95% CI 1.4–8.1) on day 2 for DHA–PQ and 79.8% (95% CI 72.3–85.7) on day 1 to 9.5% (95% CI 5.4–15.2) on day 2 for AL, (p = 0.04). There was a significant difference in the uncorrected ACPR between DHA–PQ and AL, both at 28-day and 42-day follow-up with 97.4% (95% CI 93.5–99.3) in DHA–PQ vs 84.5% (95% CI 77.8–89.8) in AL (p

Next-generation sequencing (NGS) has become a necessary tool for genomic epidemiology. Even though the utility of genomics in human health has been proved, genomic surveillance has never been as important as during the COVID-19 pandemic. This has been demonstrated by the recent use of genomic surveillance to detect new variants of SARS-CoV-2 in the United Kingdom, South Africa, and Brazil. Until recently, Malian scientists did not have access to any local NGS platform, and samples had to be shipped abroad for sequencing. Here, we report on how we adapted a laboratory setup for Plasmodium research to generate the first complete SARS-CoV-2 genome locally. Total RNA underwent a library preparation using an Illumina TruSeq stranded RNA kit. A metagenomics sequencing was performed on an Illumina MiSeq platform, which was followed by bioinformatic analyses on a local server in Mali. We recovered a full genome of SARS-CoV-2 of 29 kb with an average depth coverage of 200×. We have demonstrated our capacity to generate a high-quality genome with limited resources and highlight the need to develop genomics capacity locally to solve health problems. We discuss challenges related to access to reagents during a pandemic period and propose some home-made solutions.

BackgroundPlasmodium falciparum(Pf) whole-organism sporozoite vaccines have provided excellent protection against controlled human malaria infection (CHMI) and naturally transmitted heterogeneous Pf in the field. Initial CHMI studies showed significantly higher durable protection against homologous than heterologous strains, suggesting the presence of strain-specific vaccine-induced protection. However, interpretation of these results and understanding of their relevance to vaccine efficacy (VE) have been hampered by the lack of knowledge on genetic differences between vaccine and CHMI strains, and how these strains are related to parasites in malaria endemic regions.MethodsWhole genome sequencing using long-read (Pacific Biosciences) and short-read (Illumina) sequencing platforms was conducted to generatede novogenome assemblies for the vaccine strain, NF54, and for strains used in heterologous CHMI (7G8 from Brazil, NF166.C8 from Guinea, and NF135.C10 from Cambodia). The assemblies were used to characterize sequence polymorphisms and structural variants in each strain relative to the reference Pf 3D7 (a clone of NF54) genome. Strains were compared to each other and to clinical isolates from South America, Sub-Saharan Africa, and Southeast Asia.ResultsWhile few variants were detected between 3D7 and NF54, we identified tens of thousands of variants between NF54 and the three heterologous strains both genome-wide and within regulatory and immunologically important regions, including in pre-erythrocytic antigens that may be key for sporozoite vaccine-induced protection. Additionally, these variants directly contribute to diversity in immunologically important regions of the genomes as detected throughin silicoCD8+T cell epitope predictions. Of all heterologous strains, NF135.C10 consistently had the highest number of unique predicted epitope sequences when compared to NF54, while NF166.C8 had the lowest. Comparison to global clinical isolates revealed that these four strains are representative of their geographic region of origin despite long-term culture adaptation; of note, NF135.C10 is from an admixed population, and not part of recently-formed drug resistant subpopulations present in the Greater Mekong Sub-region.ConclusionsThese results are assisting the interpretation of VE of whole-organism vaccines against homologous and heterologous CHMI, and may be useful in informing the choice of strains for inclusion in region-specific or multi-strain vaccines.

Background Encoded by the var gene family, highly variable Plasmodium falciparum erythrocyte membrane protein-1 (PfEMP1) proteins mediate tissue-specific cytoadherence of infected erythrocytes, resulting in immune evasion and severe malaria disease. Sequencing and assembling the 40–60 var gene complement for individual infections has been notoriously difficult, impeding molecular epidemiological studies and the assessment of particular var elements as subunit vaccine candidates. Methods We developed and validated a novel algorithm, Exon-Targeted Hybrid Assembly (ETHA), to perform targeted assembly of var gene sequences, based on a combination of Pacific Biosciences and Illumina data. Results Using ETHA, we characterized the repertoire of var genes in 12 samples from uncomplicated malaria infections in children from a single Malian village and showed them to be as genetically diverse as vars from isolates from around the globe. The gene var2csa, a member of the var family associated with placental malaria pathogenesis, was present in each genome, as were vars previously associated with severe malaria. Conclusion ETHA, a tool to discover novel var sequences from clinical samples, will aid the understanding of malaria pathogenesis and inform the design of malaria vaccines based on PfEMP1. ETHA is available at: https://sourceforge.net/projects/etha/. Electronic supplementary material The online version of this article (doi:10.1186/s13073-017-0422-4) contains supplementary material, which is available to authorized users.

Previous studies have shown that a single season of intermittent preventive treatment in schoolchildren (IPTsc) targeting the transmission season has reduced the rates of clinical malaria, all-cause clinic visits, asymptomatic parasitemia, and anemia. Efficacy over the course of multiple years of IPTsc has been scantly investigated. Methods: An open, randomized-controlled trial among schoolchildren aged 6–13 years was conducted from September 2007 to January 2010 in Kolle, Mali. Students were included in three arms: sulphadoxine-pyrimethamine+artesunate (SP+AS), amodiaquine+artesunate (AQ+AS), and control (C). All students received two full doses, given 2 months apart, and were compared with respect to the incidence of clinical malaria, all-cause clinic visits, asymptomatic parasitemia, and anemia. Results: A total of 296 students were randomized. All-cause clinic visits were in the SP+AS versus control (29 (20.1%) vs. 68 (47.2%); 20 (21.7%) vs. 41 (44.6%); and 14 (21.2%) vs. 30 (44.6%); p < 0.02) in 2007, 2008, and 2009, respectively. The prevalence of asymptomatic parasitemia was lower in the SP+AS compared to control (38 (7.5%) vs. 143 (28.7%); and 47 (12.7%) vs. 75 (21.2%); p < 0.002) in 2007 and 2008, respectively. Hemoglobin concentration was significantly higher in children receiving SP+AS (11.96, 12.06, and 12.62 g/dL) than in control children (11.60, 11.64, and 12.15 g/dL; p < 0.001) in 2007, 2008, and 2009, respectively. No impact on clinical malaria was observed. Conclusion: IPTsc with SP+AS reduced the rates of all-cause clinic visits and anemia during a three-year implementation.

Malaria, an infectious disease caused by the parasite Plasmodium falciparum, causes nearly 435,000 deaths annually worldwide. RIFINs and STEVORs are two variant surface antigen families that are involved in malaria pathogenesis and immune evasion. Recent work has shown that a lack of humoral immunity to these proteins is associated with severe malaria vulnerability in Malian children. This is the first study to have compared serologic responses of children and adults to RIFINs and STEVORs in settings of malaria endemicity and to examine such serologic responses before and after a clinical malaria episode. Using microarrays, we determined that the semiconserved domains in these two parasite variant surface antigen families harbor peptides whose seroreactivity reflects malaria exposure. A similar approach has the potential to illuminate the role of variant surface antigens in the development of natural immunity to clinical malaria. Potential vaccines for severe malaria should include consideration of peptides within the semiconserved domains of RIFINs and STEVORs., The repetitive interspersed family (RIFIN) and the subtelomeric variable open reading frame (STEVOR) family represent two of three major Plasmodium falciparum variant surface antigen families involved in malaria pathogenesis and immune evasion and are potential targets in the development of natural immunity. Protein and peptide microarrays populated with RIFINs and STEVORs associated with severe malaria vulnerability in Malian children were probed with adult and pediatric sera to identify epitopes that reflect malaria exposure. Adult sera recognized and reacted with greater intensity to all STEVOR proteins than pediatric sera did. Serorecognition of and seroreactivity to peptides within the semiconserved domain of STEVORs increased with age and seasonal malaria exposure, while serorecognition and seroreactivity increased for the semiconserved and second hypervariable domains of RIFINs only with age. Serologic responses to RIFIN and STEVOR peptides within the semiconserved domains may play a role in natural immunity to severe malaria. IMPORTANCE Malaria, an infectious disease caused by the parasite Plasmodium falciparum, causes nearly 435,000 deaths annually worldwide. RIFINs and STEVORs are two variant surface antigen families that are involved in malaria pathogenesis and immune evasion. Recent work has shown that a lack of humoral immunity to these proteins is associated with severe malaria vulnerability in Malian children. This is the first study to have compared serologic responses of children and adults to RIFINs and STEVORs in settings of malaria endemicity and to examine such serologic responses before and after a clinical malaria episode. Using microarrays, we determined that the semiconserved domains in these two parasite variant surface antigen families harbor peptides whose seroreactivity reflects malaria exposure. A similar approach has the potential to illuminate the role of variant surface antigens in the development of natural immunity to clinical malaria. Potential vaccines for severe malaria should include consideration of peptides within the semiconserved domains of RIFINs and STEVORs.

Artemisinin-resistant Plasmodium falciparum malaria has been documented in southeast Asia and may already be spreading in that region. Molecular markers are important tools for monitoring the spread of antimalarial drug resistance. Recently, single-nucleotide polymorphisms (SNPs) in the PF3D7_1343700 kelch propeller (K13-propeller) domain were shown to be associated with artemisinin resistance in vivo and in vitro. The prevalence and role of K13-propeller mutations are poorly known in sub-Saharan Africa. K13-propeller mutations were genotyped by direct sequencing of nested polymerase chain reaction (PCR) amplicons from dried blood spots of pre-treatment falciparum malaria infections collected before and after the use of artemisinin-based combination therapy (ACT) as first-line therapy in Mali. Although K13-propeller mutations previously associated with delayed parasite clearance in Cambodia were not identified, 26 K13-propeller mutations were identified in both recent samples and pre-ACT infections. Parasite clearance time was comparable between infections with non-synonymous K13-propeller mutations and infections with the reference allele. These findings suggest that K13-propeller mutations are present in artemisinin-sensitive parasites and that they preceded the wide use of ACTs in Mali.

International audience; We assessed the ex vivo/in vitro sensitivity of 54 Malian Plasmodium falciparum isolates to artemisinin for the monitoring of drug resistance in this area. The artemisinin sensitivity of parasites was evaluated using 1) the ex vivo and in vitro parasite recrudescence detection after treatment of the ring stage with 1–200 nM artemisinin for 48 hours and 2) the in vitro parasite recrudescence kinetics assay over 7 days after 6-hour treatment of the ring stage with 700 nM dihydroartemisinin (DHA). In addition, as recommended by the World Health Organization for artemisinin resistance characterization, the ring-stage survival assay (RSA0–3 h) was performed and the parasite isolates were sequenced at the kelch 13 propeller locus. No clinical and molecular evidence of artemisinin resistance was observed. However, these isolates present different phenotypic profiles in response to artemisinin treatments. Despite all RSA0–3 h values less than 1.5%, six out of 46 (13.0%) isolates tested ex vivo and four out of six (66.7%) isolates tested in vitro were able to multiply after 48-hour treatments with 100 nM artemisinin. Moreover, five out of eight isolates tested showed faster parasite recovery after DHA treatment in kinetic assays. The presence of such phenotypes needs to be taken into account in the assessment of the efficacy of artemisinins in Mali. The assays presented here appear as valuable tools for the monitoring of artemisinin sensitivity in the field and thus could help to evaluate the risk of emergence of artemisinin resistance in Africa.

Background VAR2CSA, a member of the Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) family, mediates the binding of P. falciparum-infected erythrocytes to chondroitin sulfate A, a surface-associated molecule expressed in placental cells, and plays a central role in the pathogenesis of placental malaria. VAR2CSA is a target of naturally acquired immunity and, as such, is a leading vaccine candidate against placental malaria. This protein is very polymorphic and technically challenging to sequence. Published var2csa sequences, mostly limited to specific domains, have been generated through the sequencing of cloned PCR amplicons using capillary electrophoresis, a method that is both time consuming and costly, and that performs poorly when applied to clinical samples that are commonly polyclonal. A next-generation sequencing platform, Pacific Biosciences (PacBio), offers an alternative approach to overcome these issues. Methods PCR primers were designed that target a 5 kb segment in the 5′ end of var2csa and the resulting amplicons were sequenced using PacBio sequencing. The primers were optimized using two laboratory strains and were validated on DNA from 43 clinical samples, extracted from dried blood spots on filter paper or from cryopreserved P. falciparum-infected erythrocytes. Sequence reads were assembled using the SMRT-analysis ConsensusTools module. Results Here, a PacBio sequencing-based approach for recovering a segment encoding the majority of VAR2CSA’s extracellular region is described; this segment includes the totality of the first four domains in the 5′ end of var2csa (~5 kb), from clinical malaria samples. The feasibility of the method is demonstrated, showing a high success rate from cryopreserved samples and more limited success from dried blood spots stored at room temperature, and characterized the genetic variation of the var2csa locus. Conclusions This method will facilitate a detailed analysis of var2csa genetic variation and can be adapted to sequence other hypervariable P. falciparum genes. Electronic supplementary material The online version of this article (doi:10.1186/s12936-017-1976-8) contains supplementary material, which is available to authorized users.

La presente etude avait pour objectif de decrire la morbidite du paludisme et la frequence des marqueurs moleculaires de resistance a la sulfadoxine-pyrimethamine (SP) et a la chloroquine (CQ) dans une zone d’endemie palustre au Mali. Il s’agissait d’une etude transversale d’observation effectuee de novembre 2005 a janvier 2006. L’etude a porte sur 200 femmes enceintes ayant accouche a la maternite de Fana, parmi lesquelles la classe d’âge des 14–19 ans (28,5 %) predominait. Au total, 52,3 % (104/200) et 15 % (30/200) des femmes examinees etaient porteuses de Plasmodium falciparum respectivement dans le sang peripherique et le placenta. Le taux de prevalence de l’anemie etait de 44,5 % (89/200). La frequence de la mutation Pfcrt K76T de l’infection due a P. falciparum dans le sang peripherique etait de 68,8 % (11/16) contre 100 %(16/16) dans le placenta (p = 0,004). La mutation DHFR N51I etait presente dans 12,5 % (2/16) et 18,8 % (3/16) des cas respectivement dans le sang peripherique et dans le placenta (p= 0,12). Quant a la mutation DHPS A437G, elle a ete observee avec une frequence de 25 % (4/16) aussi bien dans le sang peripherique que dans le placenta. En revanche, nous n’avons pas mis en evidence de mutants DHPS K540E et DHFR 164L.

Resume But Il est classiquement admis que le paludisme neonatal est un evenement rare en zone d’endemie palustre. De plus en plus d’etudes prouvent une sous-estimation de cette frequence. Notre travail avait pour objectif de determiner la prevalence du paludisme congenital et neonatal dans une structure de niveau tertiaire de reference nationale pour la prise en charge des nouveau-nes au Mali en utilisant trois methodes complementaires de diagnostic. Materiel et methodes Le travail s’est deroule a l’unite de neonatologie du service de pediatrie du centre hospitalier universitaire de Gabriel-Toure, Bamako, Mali du 1er octobre 2006 au 17 mars 2008. Ont ete inclus tous les nouveau-nes a terme de 0 a 28 jours hospitalises dans le service ainsi que leurs meres. Pour le diagnostic du paludisme, un test de diagnostic rapide (TDR, OptiMAL IT®), une goutte epaisse et une reaction de polymerisation en chaine (PCR) ont ete realises sur un prelevement veineux de la mere et du nouveau-ne. Resultats Nous avons inclus 267 nouveau-nes et 146 meres. L’âge moyen des nouveau-nes etait de 2,7 jours et le poids moyen de 2862,4 g. La microscopie et la PCR sont revenus negatifs chez tous les nouveau-nes. Les meres etaient âgees de 25 ans en moyenne. La goutte epaisse est revenue negative chez toutes les meres, le TDR positif a Plasmodium falciparum chez une mere et la PCR positive pour neuf meres (sept cas de P. falciparum et deux cas de Plasmodium ovale). Conclusion Le paludisme congenital et neonatal demeure un evenement rare chez le nouveau-ne admis dans une structure de reference tertiaire au Mali.

Contains fulltext : 89111.pdf (Publisher’s version ) (Closed access) Sulfadoxine-pyrimethamine (SP) is currently the drug of choice for intermittent preventive treatment of Plasmodium falciparum both in pregnancy and infancy. A prolonged parasite clearance time conferred by dhfr and dhps mutations is believed to be responsible for increased gametocyte prevalence in SP treated individuals. However, using a direct feeding assay in Mali, we showed that gametocytes present in peripheral venous blood post-SP treatment had reduced infectivity for Anopheles gambiae sensu stricto (ss) mosquitoes. We investigated the potential mechanisms involved in the dhfr and dhps quintuple mutant NF-135 and the single dhps 437 mutant NF-54. Concentrations of sulfadoxine (S) and pyrimethamine (P) equivalent to the serum levels of the respective drugs on day 3 (S=61 microg/ml, P=154.7 ng/ml) day 7 (S=33.8 microg/ml, P=66.6 ng/ml) and day 14 (S=14.2 microg/ml, P=15.7 ng/ml) post-SP treatment were used to study the effect on gametocytogenesis, gametocyte maturation and infectivity to Anopheles stephensi mosquitoes fed through an artificial membrane. The drugs readily induced gametocytogenesis in the mutant NF-135 strain but effectively killed the wild-type NF-54. However, both drugs impaired gametocyte maturation yielding odd-shaped non-exflagellating mature gametocytes. The concomitant ingestion of both S and P together with gametocytemic blood-meal significantly reduced the prevalence of oocyst positivity as well as oocyst density when compared to controls (P

Plasmodium falciparum chloroquine resistance (CQR) transporter point mutation (PfCRT 76T) is known to be the key determinant of CQR. Molecular detection of PfCRT 76T in field samples may be used for the surveillance of CQR in malaria-endemic countries. The genotype-resistance index (GRI), which is obtained as the ratio of the prevalence of PfCRT 76T to the incidence of CQR in a clinical trial, was proposed as a simple and practical molecular-based addition to the tools currently available for monitoring CQR in the field. In order to validate the GRI model across populations, time, and resistance patterns, we compiled data from the literature and generated new data from 12 sites across Mali. We found a mean PfCRT 76T mutation prevalence of 84.5% (range 60.9-95.1%) across all sites. CQR rates predicted from the GRI model were extrapolated onto a map of Mali to show the patterns of resistance throughout the participating regions. We present a comprehensive map of CQR in Mali, which strongly supports recent changes in drug policy away from chloroquine.

Background The host response to infection by Plasmodium falciparum, the parasite most often responsible for severe malaria, ranges from asymptomatic parasitaemia to death. The clinical trajectory of malaria is influenced by host genetics and parasite load, but the factors determining why some infections produce uncomplicated malaria and some proceed to severe disease remain incompletely understood. Methods To identify molecular markers of severe falciparum malaria, human gene expression patterns were compared between children aged 6 months to 5 years with severe and uncomplicated malaria who were enrolled in a case–control study in Bandiagara, Mali. Microarrays were used to obtain expression data on severe cases and uncomplicated controls at the time of acute disease presentation (five uncomplicated and five severe), 1 week after presentation (three uncomplicated and three severe) and treatment initiation, and in the subsequent dry season (late convalescence, four uncomplicated and four severe). This is a pilot study for the first use of microarray technology in Mali. Results Complement and toll-like receptor (TLR) pathways were differentially expressed, with severe cases showing higher expression of the C1q, TLR2, TLR4, TLR8, and CR1 genes. Other genes previously associated with malaria pathogenesis, GZMB, FOS and HSPA6, were also higher among severe cases. TLR2, TLR4, TLR8, CR1, GZMB, FOS, and HSPA6 genes were expressed at lower levels in severe cases at late convalescence. Conclusions Overexpression of genes previously associated with uncomplicated malaria was associated with severe disease. Low baseline expression of these genes may represent candidate markers for severe malaria. Despite the small sample size, results of this pilot study offer promising targets for follow-up analyses. Electronic supplementary material The online version of this article (doi:10.1186/s12936-016-1189-6) contains supplementary material, which is available to authorized users.

International audience; Although artemisinin resistance has yet to be reported in Africa, surveillance of the efficacy of artemisinin-based combination therapies (ACTs) is warranted. Here, the efficacy of artesunate + sulfadoxine-pyrimethamine (AS+SP) and artemether-lumefantrine (AL) was evaluated in Mali. Randomized open-label comparative in vivo assay of AS+SP versus AL were carried out using the 28-day follow-up World Health Organization protocol. Patients with uncomplicated falciparum malaria and at least 6 months of age were recruited between October 2010 and January 2014. A subset of these patients was selected to measure Plasmodium falciparum clearance time. Polymerase chain reaction corrected adequate clinical and parasitological responses were 100% for AS+SP and 98.2% for AL with no significant difference (P = 0.06). The reinfection rates were comparable (P = 0.63) with 8.0% for AS+SP and 12.6% for AL. Individuals under 8 years were more susceptible to treatment failure (relative risk = 1.9; 95% confidence interval = 1.2, 3.3). Median parasite clearance half-life was 1.7 hours (interquartile range [IQR] = 1.3-2.2) for AS+SP and 1.9 hours (IQR = 1.5-2.5) for AL with no statistically significant difference (P = 0.24). Efficacy of AS+SP and AL was high. This study provides baseline information on parasite clearance half-lives after ACT treatment, particularly AS+SP, in Mali.

Background Plasmodium falciparum resistance to artemisinin has been reported in South-East Asia. Long half-life drugs are increasingly being used for malaria prevention. The potential spread of parasite resistance to these regimens is real and makes regular efficacy surveillance a priority. Methods From August to December 2004 and July to December 2005, a randomized open label trial of sulphadoxine-pyrimethamine (SP) + artesunate (AS) versus SP + amodiaquine (AQ), and SP alone, was conducted in two villages of Mali. PCR was used to distinguish new infections from recrudescent P. falciparum infections. Patients were followed for 28 days to assess treatment efficacy. Results Overall 912 children aged between six to 59 months, with uncomplicated P. falciparum malaria were recruited. Baseline characteristics were similar in the three treatment arms. Crude ACPRs were 94.9%; 98.6% and 93.5% for SP + AS; SP + AQ and SP alone arms respectively (SP + AS versus SP + AQ, p = 0.01; SP + AS versus SP, p = 0.5; SP + AQ versus SP, p = 0.001). After PCR adjustment, cACPRs were 99%; 100% and 97.2% for SP + AS; SP + AQ and SP alone arms, respectively (SP + AS versus SP + AQ, p = 0.25; SP + AS versus SP, p = 0.12; SP + AQ versus SP, p = 0.007). Conclusion Sulphadoxine-pyrimethamine + amodiaquine therapy was as efficacious as sulphadoxine-pyrimethamine + artesunate, but more efficacious than sulphadoxine-pyrimethamine alone in the treatment of uncomplicated P. falciparum malaria in Mali.

Background Artemisinin-based combination therapies (ACTs) are the main treatment for malaria in endemic countries. Plasmodium falciparum resistance to artemisinins is described as delayed parasite clearance, which is associated with mutations on the parasite K13 propeller gene. Both the mechanisms of action and mechanisms of resistance to artemisinins are poorly understood. Transcriptomic studies can help in improving our understanding of these processes. Here we explore P. falciparum in vivo RNA expression profile after a curative dose of artesunate monotherapy. Methods During a prospective study of the efficacy of artesunate in monotherapy in children aged 1–10 years and presenting uncomplicated P. falciparum malaria in Bougoula-Hameau, Mali, venous blood was collected on PAXgen blood RNA tubes before treatment (H0) and one (H1), two (H2) and three hours (H3) after treatment. RNA was extracted from these respective blood samples and used for microarray experiments with Plasmodium/Anopheles GeneChips and the Affymetrix® platform. Results A total of 23 samples from 6 patients were included in the final analysis after quality control using Affimetrix® and Qlucore® softwares. With a 2-groups comparison of H0/H after treatment, 236 genes were identified as differentially expressed. Overall 42 genes were up-regulated including a knob-associated histidine-rich protein, rifins (pf.12.409.0, pf.13_399.0), stevors (pf.3.184.0), RESA-like proteins with DNAJ domain and thioredoxins. Heat shock protein (Pf.5.258.0), a number of AP2 domain-containing genes (Pf.6.27.0, Pf.11.99.0), an ABC transporter (Pf.12.250.0), genes involved in cell cycle regulation and many exported protein genes with unknown function and membrane proteins genes were among the 194 down-regulated genes. Conclusions Our data support a role for these genes in the in vivo response of P. falciparum to artesunate administration.

Control of uncomplicated cases of malaria has been greatly impaired by development of resistance against mainstay antimalarial agents [1, 2]. The first–line antimalarial strategy is presently artemisinin combination therapy. Quinine (QN) is used in many malaria-endemic countries for the management of severe life–threatening malaria and as rescue therapy for parasites resistant to other antimalarial drugs. As a monotherapy, QN is associated with significant recrudescence rates [3]. This may be because QN is a rapidly acting drug with a relatively short elimination half–life [4, 5]. Failure of QN in malaria treatment has been long documented, particularly in South America [6, 7] and Southeast Asia [8]. Although high–level resistance of Plasmodium falciparum to QN is rare, in vivo and in vitro decreases in parasite susceptibility have led to the use of this drug in combination, particularly with the antibiotics tetracycline or doxycycline [9–13]. The mechanisms of decreased P. falciparum susceptibility to QN are not clearly understood. Modulation of in vitro QN sensitivity has been associated with single nucleotide polymorphisms in the pfcrt (chloroquine–resistance transporter) and pfmdr1 (multidrug resistance 1) genes [14, 15]. Involvement of additional genes also seems to be important, because the association of pfcrt and pfmdr1 is not always observed [16]. This reality of a complex phenotype is also supported by the frequent efficacy of QN against chloroquine-resistant parasites [17, 18]. The availability of a laboratory-derived genetic cross between 2 parasites of markedly different in vitro QN responses [19] permitted a quantitative trait loci–based approach that identified genomic regions contributing to the phenotypes [18]. Analysis showed a possible association between reduced P. falciparum susceptibility to QN and a locus on chromosome 13. A pairwise effect was also detected between that chromosome 13 locus and an additional locus on chromosome 9. The set of predicted genes on the chromosome 13 locus included a homologue to the family of Na+/H+ exchanger (NHE) proteins [18]. The P. falciparum–encoded PfNHE–1 is 1920 amino acids long and harbors an open reading frame with a microsatellite polymorphism consisting of variable DNNND repeat units, designated ms4760. The predicted pfnhe–1 sequence was analyzed in the Dd2 × HB3 progeny, as well as in a range of field isolates and laboratory strains with variable susceptibilities to QN. Eight variants of ms4760 were originally discovered. The variant ms4760–1 with 2 copies of DNNND repeat units was significantly associated with reduced in vitro QN sensitivity [18]. These results were later supported by transfection experiments [20]. The aforementioned studies were performed with in vitro cultured laboratory strains. However, these polymorphisms were also present in natural P. falciparum populations [21] and found to be associated with QN sensitivity among field isolates in in vitro assays [22–24]. Here we extend these studies to clinical settings by exploring the in vivo impact of QN therapy on the prevalence of the pfnhe–1 ms4760 and pfcrt polymorphisms in 2 Malian villages.

Plasmodium falciparum resistance to artemisinins by delayed parasite clearance is present in Southeast Asia. Scant data on parasite clearance after artemisinins are available from Africa, where transmission is high, burden is greatest, and artemisinin use is being scaled up. Children 1–10 years of age with uncomplicated malaria were treated with 7 days of artesunate and followed for 28 days. Blood smears were done every 8 hours until negative by light microscopy. Results were compared with a similar study conducted in the same village in 2002–2004. The polymerase chain reaction-corrected cure rate was 100%, identical to 2002–2004. By 24 hours after treatment initiation, 37.0% of participants had cleared parasitemia, compared with 31.9% in 2002–2004 (P = 0.5). The median parasite clearance time was 32 hours. Only one participant still had parasites at 48 hours and no participant presented parasitemia at 72 hours. Artesunate was highly efficacious, with no evidence of delayed parasite clearance. We provide baseline surveillance data for the emergence or dissemination of P. falciparum resistance in sub-Saharan Africa.

In 2003, Mali introduced intermittent preventive therapy in pregnancy (ITPp) with sulfadoxine-pyrimethamine (SP) for the control of malaria in pregnancy, consisting of 2 doses of SP given in the 2nd and 3rd trimester. This widely used regimen, although very effective, leaves many women unprotected from malaria during the last 4-to-8 weeks of gestation, which is a pivotal period for fetal weight gain. The aim of the study was to compare the efficacy and safety of 3-dose versus 2-dose IPTp-SP for the prevention of placental malaria and associated low birth weight (LBW). We conducted a parallel-group, open-label, individually randomized controlled superiority trial involving 814 women of all gravidity, enrolled from April 2006 through March 2008. All women were seen at least 3 times and received either 2 (n = 401) or 3 (n = 413) doses of IPTp-SP. The primary endpoint measured was placental malaria, LBW, preterm births, and maternal anemia were secondary endpoints, and severe maternal skin reactions and neonatal jaundice were safety endpoints. Among the 96% of study subjects who were followed up until delivery, the prevalence of placental malaria was 2-fold lower in the 3-dose group (8.0%) than in the 2-dose group (16.7%); the adjusted prevalence ratio (APR) was 0.48 (95% confidence interval [CI], 0.32-0.71). LBW and preterm births were also reduced; the prevalence of LBW was 6.6% in the 3-dose group versus 13.3% in the 2-dose group (APR, 0.50; 95% CI, 0.32-0.79), and the prevalence of preterm births was 3.2% versus 8.9% (APR, 0.37; 95% CI, 0.19-0.71). No significant reductions in maternal anemia or differences in safety endpoints were observed. Adding a third dose of ITPp-SP halved the risk of placental malaria, LBW, and preterm births in all gravidae, compared with the standard 2-dose regimen, in this area of highly seasonal transmission with low levels of SP resistance. Clinical Trials Registration: ISRCTN 74189211