Randomized Field Trial to Assess the Safety and Efficacy of Dihydroartemisinin-Piperaquine for Seasonal Malaria Chemoprevention in School-Aged Children in Bandiagara, Mali.

BACKGROUND: Owing to the increased cases of malaria in older children, the World Health Organization has recently recommended extending seasonal malaria chemoprevention (SMC) to children >5 years of age and using other effective drugs for malaria. In this study, we report the safety and efficacy of dihydroartemisinin-piperaquine (DHA-PQ) for SMC in school-aged children in Mali. METHOD: This randomized, controlled trial included 345 participants aged 6-15 years randomized to receive DHA-PQ, sulfadoxine-pyrimethamine plus amodiaquine (SP-AQ), or no chemoprevention (albendazole) at a 1:1:1 ratio. Four rounds of SMC were conducted from September to December 2021. The participants were assessed 7 days after each round for safety and efficacy of the interventions. RESULTS: Abdominal pain (11.8% vs 29.2%), headache (11.2% vs 19.2%), and vomiting (5.7% vs 15.2%) were frequently reported in the DHA-PQ and SP-AQ arms. On Day 120 of follow up, the incidence of clinical malaria was 0.01 episodes/person-month in the DHA-PQ and SP-AQ arms and 0.17 episodes/person-month in the control arm (P < .0001). Gametocytes were detected in 37 participants in all arms. CONCLUSIONS: Children in DHA-PQ arm reported less adverse events compared to the SP-AQ arm. Both drugs were effective against clinical malaria and infection.

IFNγ, TNFα polymorphisms and IFNγ serum levels are associated with the clearance of drug-resistant P. falciparum in Malian children.

Host immunity has been suggested to clear drug-resistant parasites in malaria-endemic settings. However, the immunogenetic mechanisms involved in parasite clearance are poorly understood. Characterizing the host's immunity and genes involved in controlling the parasitic infection can inform the development of blood-stage malaria vaccines. This study investigates host regulatory cytokines and immunogenomic factors associated with the clearance of Plasmodium falciparum carrying a chloroquine resistance genotype. Biological samples from participants of previous drug efficacy trials conducted in two Malian localities were retrieved. The P. falciparum chloroquine resistance transporter (Pfcrt) gene was genotyped using parasite DNA. Children carrying parasites with the mutant allele (Pfcrt-76T) were classified based on their ability to clear their parasites. The levels of the different cytokines were measured in serum. The polymorphisms of specific human genes involved in malaria susceptibility were genotyped using human DNA. The prevalence of the Pfcrt-76T was significantly higher in Kolle than in Bandiagara (81.6 % vs 38.6 %, p < 10-6). The prevalence of children who cleared their mutant parasites was significantly higher in Bandiagara than in Kolle (82.2 % vs 67.4 %, p < 0.05). The genotyping of host genes revealed that IFN-γ -874 T and TNF-α -308A alleles were positively associated with parasite clearance. Cytokine profiling revealed that IFN-γ level was positively associated with parasite clearance (p = 0.04). This study highlights the role of host's immunity and immunogenetic factors to clear resistant parasites, suggesting further characterization of these polymorphisms may help to develop novel approaches to antiparasitic treatment strategies.

Selection of pfcrt K76 and pfmdr1 N86 Coding Alleles after Uncomplicated Malaria Treatment by Artemether-Lumefantrine in Mali.

BACKGROUND: Artemether-lumefantrine is a highly effective artemisinin-based combination therapy that was adopted in Mali as first-line treatment for uncomplicated Plasmodium falciparum malaria. This study was designed to measure the efficacy of artemether-lumefantrine and to assess the selection of the P. falciparum chloroquine resistance transporter (pfcrt) and P. falciparum multi-drug resistance 1 (pfmdr1) genotypes that have been associated with drug resistance. METHODS: A 28-day follow-up efficacy trial of artemether-lumefantrine was conducted in patients aged 6 months and older suffering from uncomplicated falciparum malaria in four different Malian areas during the 2009 malaria transmission season. The polymorphic genetic markers MSP2, MSP1, and Ca1 were used to distinguish between recrudescence and reinfection. Reinfection and recrudescence were then grouped as recurrent infections and analyzed together by PCR-restriction fragment length polymorphism (RFLP) to identify candidate markers for artemether-lumefantrine tolerance in the P. falciparum chloroquine resistance transporter (pfcrt) gene and the P. falciparum multi-drug resistance 1 (pfmdr1) gene. RESULTS: Clinical outcomes in 326 patients (96.7%) were analyzed and the 28-day uncorrected adequate clinical and parasitological response (ACPR) rate was 73.9%. The total PCR-corrected 28-day ACPR was 97.2%. The pfcrt 76T and pfmdr1 86Y population prevalence decreased from 49.3% and 11.0% at baseline (n = 337) to 38.8% and 0% in patients with recurrent infection (n = 85); p = 0.001), respectively. CONCLUSION: Parasite populations exposed to artemether-lumefantrine in this study were selected toward chloroquine-sensitivity and showed a promising trend that may warrant future targeted reintroduction of chloroquine or/and amodiaquine.

Effect of routine seasonal malaria chemoprevention on malaria trends in children under 5 years in Dangassa, Mali.

BACKGROUND: Seasonal malaria chemoprevention (SMC) is a new strategy to prevent malaria in children under 5 years old. It has been recommended by the World Health Organization since 2012 in malaria-endemic areas with seasonal transmission. This study aimed to assess the changes in malaria indicators through two consecutive years of SMC routine implementation in children under 5 years old in Dangassa, where malaria is endemic with a long and high transmission season. METHODS: From 2012 to 2016, a cohort study was conducted in Dangassa village. The study team based in the village followed all malaria clinical cases in children under 5 years old at the community health centre. During the study, SMC was routinely implemented in collaboration with the National Malaria Control Programme. The Cox regression model was used in order to compare malaria risk during the study. RESULTS: The Cox regression model showed a significant reduction in malaria clinical incidence, both in 2015 (HR = 0.27 (0.18-0.40), 95% CI) and in 2016 (HR = 0.23 (0.15-0.35), 95% CI) of SMC implementation compared to October 2013. Gametocyte and fever prevalence was lower between September and October during SMC implementation (2015 and 2016) compared to the same period before SMC implementation (2013-2014). A slight increase of malaria incidence was observed in December at the end of SMC implementation. CONCLUSION: SMC has significantly reduced both malaria incidence and gametocyte prevalence and improved haemoglobin levels in children under 5 years old after 2 years of routine implementation.

A comprehensive analysis of drug resistance molecular markers and Plasmodium falciparum genetic diversity in two malaria endemic sites in Mali.

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.

Immunoglobulin G subclass and antibody avidity responses in Malian children immunized with Plasmodium falciparum apical membrane antigen 1 vaccine candidate FMP2.1/AS02A.

BACKGROUND: A malaria vaccine based on Plasmodium falciparum apical membrane antigen 1 (AMA1) elicited strain specific efficacy in Malian children that waned in the second season after vaccination despite sustained AMA1 antibody titers. With the goal of identifying a humoral correlate of vaccine-induced protection, pre- and post-vaccination sera from children vaccinated with the AMA1 vaccine and from a control group that received a rabies vaccine were tested for AMA1-specific immunoglobulin G (IgG) subclasses (IgG1, IgG2, IgG3, and IgG4) and for antibody avidity. METHODS: Samples from a previously completed Phase 2 AMA1 vaccine trial in children residing in Mali, West Africa were used to determine AMA1-specific IgG subclass antibody titers and avidity by ELISA. Cox proportional hazards models were used to assess correlation between IgG subclass antibody titers and risk of time to first or only clinical malaria episode and risk of multiple episodes. Asexual P. falciparum parasite density measured for each child as area under the curve were used to assess correlation between IgG subclass antibody titers and parasite burden. RESULTS: AMA1 vaccination did not elicit a change in antibody avidity; however, AMA1 vaccinees had a robust IgG subclass response that persisted over the malaria transmission season. AMA1-specific IgG subclass responses were not associated with decreased risk of subsequent clinical malaria. For the AMA1 vaccine group, IgG3 levels at study day 90 correlated with high parasite burden during days 90-240. In the control group, AMA1-specific IgG subclass rise and persistence over the malaria season was modest and correlated with age. In the control group, titers of several IgG subclasses at days 90 and 240 correlated with parasite burden over the first 90 study days, and IgG3 at day 240 correlated with parasite burden during days 90-240. CONCLUSIONS: Neither IgG subclass nor avidity was associated with the modest, strain-specific efficacy elicited by this blood stage malaria vaccine. Although a correlate of protection was not identified, correlations between subclass titers and age, and correlations between IgG subclass titers and parasite burden, defined by area under the curve parasitaemia levels, were observed, which expand knowledge about IgG subclass responses. IgG3, known to have the shortest half-life of the IgG subclasses, might be the most temporally relevant indicator of ongoing malaria exposure when examining antibody responses to AMA1.

Stage-dependent fate of Plasmodium falciparum-infected red blood cells in the spleen and sickle-cell trait-related protection against malaria.

BACKGROUND: Sickle-cell trait (HbAS) reduces falciparum malaria risk and suppresses parasitaemia. Although several candidate mechanisms have been proposed, their epidemiological, clinical and experimental correlates have not been adequately explained. To explore the basis for generally lower parasitaemias and delayed malaria episodes in children with HbAS, it is hypothesized here that their spleen-dependent removal of ring-infected red blood cells (RBCs) is more efficient than in children with normal haemoglobin A (HbAA). METHODS: The mechanical splenic retention of Plasmodium falciparum-infected RBCs from subjects with HbAS or HbAA was investigated using two physiologically relevant methods: microsphiltration and ex vivo spleen perfusion. P. falciparum-infected RBCs obtained from in vitro cultures and from patients were used in either normoxic or hypoxic conditions. The effect of sickling in ring-infected HbAS RBCs was also investigated. RESULTS: When a laboratory-adapted parasite strain was analysed, ring-infected HbAA RBCs were retained in microsphilters at similar or greater levels than ring-infected HbAS RBCs, under normoxic (retention rate 62.5 vs 43.8 %, P < 0.01) and hypoxic (54.0 vs 38.0 %, P = 0.11) conditions. When parasitized RBCs from Malian children were analysed, retention of ring-infected HbAA and HbAS RBCs was similar when tested either directly ex vivo (32.1 vs 28.7 %, P = 0.52) or after one re-invasion in vitro (55.9 vs 43.7 %, P = 0.30). In hypoxia, sickling of uninfected and ring-infected HbAS RBCs (8.6 vs 5.7 %, P = 0.51), and retention of ring-infected HbAA and HbAS RBCs in microsphilters (72.5 vs 68.8 %, P = 0.38) and spleens (41.2 vs 30.4 %, P = 0.11), also did not differ. Retention of HbAS and HbAA RBCs infected with mature P. falciparum stages was greater than 95 %. CONCLUSIONS: Sickle-cell trait is not associated with higher retention or sickling of ring-infected RBCs in experimental systems reflecting the mechanical sensing of RBCs by the human spleen. As observed with HbAA RBCs, HbAS RBCs infected with mature parasites are completely retained. Because the cytoadherence of HbAS RBCs infected with mature parasites is impaired, the very efficient splenic retention of such non-adherent infected RBCs is expected to result in a slower rise of P. falciparum parasitaemia in sickle-cell trait carriers.

Phase 1 randomized controlled trial to evaluate the safety and immunogenicity of recombinant Pichia pastoris-expressed Plasmodium falciparum apical membrane antigen 1 (PfAMA1-FVO [25-545]) in healthy Malian adults in Bandiagara.

BACKGROUND: The safety and immunogenicity of PfAMA1, adjuvanted with Alhydrogel(®) was assessed in malaria-experienced Malian adults. The malaria vaccine, PfAMA1-FVO [25-545] is a recombinant protein Pichia pastoris-expressed AMA-1 from Plasmodium falciparum FVO clone adsorbed to Alhydrogel(®), the control vaccine was tetanus toxoid produced from formaldehyde detoxified and purified tetanus toxin. METHODS: A double blind randomized controlled phase 1 study enrolled and followed 40 healthy adults aged 18-55 years in Bandiagara, Mali, West Africa, a rural setting with intense seasonal transmission of P. falciparum malaria. Volunteers were randomized to receive either 50 µg of malaria vaccine or the control vaccine. Three doses of vaccine were given on Days 0, 28 and 56, and participants were followed for 1 year. Solicited symptoms were assessed for seven days and unsolicited symptoms for 28 days after each vaccination. Serious adverse events were assessed throughout the study. The titres of anti-AMA-1 antibodies were measured by ELISA and P. falciparum growth inhibition assays were performed. RESULTS: Commonest local solicited adverse events were the injection site pain and swelling more frequent in the PfAMA1 group. No vaccine related serious adverse events were reported. A significant 3.5-fold increase of anti-AMA-1 IgG antibodies was observed in malaria vaccine recipients four weeks after the third immunization compared to the control group. CONCLUSION: The PfAMA1 showed a good safety profile. Most adverse events reported were of mild to moderate intensity. In addition, the vaccine induced a significant though short-lived increase in the anti-AMA1 IgG titres. Registered on www.clinicaltrials.gov with the number NCT00431808.

Hemoglobin C Trait Provides Protection From Clinical Falciparum Malaria in Malian Children.

BACKGROUND: Hemoglobin C trait, like hemoglobin S trait, protects against severe malaria in children, but it is unclear whether hemoglobin C trait also protects against uncomplicated malaria. We hypothesized that Malian children with hemoglobin C trait would have a lower risk of clinical malaria than children with hemoglobin AA. METHODS: Three hundred children aged 0-6 years were enrolled in a cohort study of malaria incidence in Bandiagara, Mali, with continuous passive and monthly active follow-up from June 2009 to June 2010. RESULTS: Compared to hemoglobin AA children (n = 242), hemoglobin AC children (n = 39) had a longer time to first clinical malaria episode (hazard ratio [HR], 0.19; P = .001; 364 median malaria-free days vs 181 days), fewer episodes of clinical malaria, and a lower cumulative parasite burden. Similarly, hemoglobin AS children (n = 14) had a longer time to first clinical malaria episode than hemoglobin AA children (HR, 0.15; P = .015; 364 median malaria-free days vs 181 days), but experienced the most asymptomatic malaria infections of any group. CONCLUSIONS: Both hemoglobin C and S traits exerted a protective effect against clinical malaria episodes, but appeared to do so by mechanisms that differentially affect the response to infecting malaria parasites.

Drying anti-malarial drugs in vitro tests to outsource SYBR green assays.

BACKGROUND: Measurement of anti-malarial drug efficacy and resistance relies mainly on in vivo clinical trials, in vitro/ex vivo assays and molecular markers detection. The existing in vitro/ex vivo assays, in particular those that are using non-radioactive devices, need to be standardized and adapted to field conditions. SYBR Green assay offers a rapid and cheap alternative to other in vitro assays, but it requires tools not commonly available in field laboratories. Here is described a modified SYBR green I protocol to perform the parasite growth test with blood samples in endemic areas, followed later by the SYBR green fluorescence assay performed at a specialized laboratory level. METHODS: In vitro susceptibility of Plasmodium falciparum clones HB3, 3D7, W2 and 7G8 to chloroquine (CQ), dihydroartemisinin (DHA), pyronaridine (PYD) and piperaquine (PPQ) was tested. Fresh isolates of P. falciparum from imported malaria cases were collected for ex vivo assays. The parasite suspension was added in 96-well plates predosed with anti-malarial drugs and incubated for 72 hours at 37°C, 5% CO2. SYBR green I protocol was modified to dry the plates after freeze-thawed process to mimic storage and shipping conditions. The plates were rehydrated with 200 µl of complete RPMI medium for fluorescence assay. RESULTS: There were no significant differences in IC50 values of CQ, DHA, PYD and PPQ, determined by the modified protocol, compared to standard protocol. Longer storage did not affect the IC50 values. CONCLUSION: The SYBR green I modified protocol produced reliable results and could be a suitable method for in vitro/ex vivo assays in field.

Understandings of genomic research in developing countries: a qualitative study of the views of MalariaGEN participants in Mali.

BACKGROUND: Obtaining informed consent for participation in genomic research in low-income settings presents specific ethical issues requiring attention. These include the challenges that arise when providing information about unfamiliar and technical research methods, the implications of complicated infrastructure and data sharing requirements, and the potential consequences of future research with samples and data. This study investigated researchers' and participants' parents' experiences of a consent process and understandings of a genome-wide association study of malaria involving children aged five and under in Mali. It aimed to inform best practices in recruiting participants into genomic research. METHODS: A qualitative rapid ethical assessment was undertaken. Fifty-five semi-structured interviews were conducted with the parents of research participants. An additional nine semi-structured interviews were conducted with senior research scientists, research assistants and with a member of an ethics committee. A focus group with five parents of research participants and direct observations of four consent processes were also conducted. French and translated English transcripts were descriptively and thematically coded using OpenCode software. RESULTS: Participants' parents in the MalariaGEN study had differing understandings of the causes of malaria, the rationale for collecting blood samples, the purposes of the study and the kinds of information the study would generate. Genomic aspects of the research, including the gene/environment interaction underlying susceptibility or resistance to severe malaria, proved particularly challenging to explain and understand. CONCLUSIONS: This study identifies a number of areas to be addressed in the design of consent processes for genomic research, some of which require careful ethical analysis. These include determining how much information should be provided about differing aspects of the research and how best to promote understandings of genomic research. We conclude that it is important to build capacity in the design and conduct of effective and appropriate consent processes for genomic research in low and middle-income settings. Additionally, consideration should be given to the role of review committees and community consultation activities in protecting the interests of participants in genomic research.

A field trial to assess a blood-stage malaria vaccine.

BACKGROUND: Blood-stage malaria vaccines are intended to prevent clinical disease. The malaria vaccine FMP2.1/AS02(A), a recombinant protein based on apical membrane antigen 1 (AMA1) from the 3D7 strain of Plasmodium falciparum, has previously been shown to have immunogenicity and acceptable safety in Malian adults and children. METHODS: In a double-blind, randomized trial, we immunized 400 Malian children with either the malaria vaccine or a control (rabies) vaccine and followed them for 6 months. The primary end point was clinical malaria, defined as fever and at least 2500 parasites per cubic millimeter of blood. A secondary end point was clinical malaria caused by parasites with the AMA1 DNA sequence found in the vaccine strain. RESULTS: The cumulative incidence of the primary end point was 48.4% in the malaria-vaccine group and 54.4% in the control group; efficacy against the primary end point was 17.4% (hazard ratio for the primary end point, 0.83; 95% confidence interval [CI], 0.63 to 1.09; P=0.18). Efficacy against the first and subsequent episodes of clinical malaria, as defined on the basis of various parasite-density thresholds, was approximately 20%. Efficacy against clinical malaria caused by parasites with AMA1 corresponding to that of the vaccine strain was 64.3% (hazard ratio, 0.36; 95% CI, 0.08 to 0.86; P=0.03). Local reactions and fever after vaccination were more frequent with the malaria vaccine. CONCLUSIONS: On the basis of the primary end point, the malaria vaccine did not provide significant protection against clinical malaria, but on the basis of secondary results, it may have strain-specific efficacy. If this finding is confirmed, AMA1 might be useful in a multicomponent malaria vaccine. (Funded by the National Institute of Allergy and Infectious Diseases and others; ClinicalTrials.gov number, NCT00460525.).

Stable malaria incidence despite scaling up control strategies in a malaria vaccine-testing site in Mali.

BACKGROUND: The recent decline in malaria incidence in many African countries has been attributed to the provision of prompt and effective anti-malarial treatment using artemisinin-based combination therapy (ACT) and to the widespread distribution of long-lasting, insecticide-treated bed nets (LLINs). At a malaria vaccine-testing site in Bandiagara, Mali, ACT was introduced in 2004, and LLINs have been distributed free of charge since 2007 to infants after they complete the Expanded Programme of Immunization (EPI) schedule and to pregnant women receiving antenatal care. These strategies may have an impact on malaria incidence. METHODS: To document malaria incidence, a cohort of 400 children aged 0 to 14 years was followed for three to four years up to July 2013. Monthly cross-sectional surveys were done to measure the prevalence of malaria infection and anaemia. Clinical disease was measured both actively and passively through continuous availability of primary medical care. Measured outcomes included asymptomatic Plasmodium infection, anaemia and clinical malaria episodes. RESULTS: The incidence rate of clinical malaria varied significantly from June 2009 to July 2013 without a clear downward trend. A sharp seasonality in malaria illness incidence was observed with higher clinical malaria incidence rates during the rainy season. Parasite and anaemia point prevalence also showed seasonal variation with much higher prevalence rates during rainy seasons compared to dry seasons. CONCLUSIONS: Despite the scaling up of malaria prevention and treatment, including the widespread use of bed nets, better diagnosis and wider availability of ACT, malaria incidence did not decrease in Bandiagara during the study period.

Adoption potential of conservation agriculture practices in sub-Saharan Africa: results from five case studies.

Despite the reported benefits of conservation agriculture (CA), its wider up-scaling in Sub-Saharan Africa (SSA) has remained fairly limited. This paper shows how a newly developed qualitative expert assessment approach for CA adoption (QAToCA) was applied to determine its adoption potential in SSA. CA adoption potential is not a predictor of observed adoption rates. Instead, our aim was to systematically check relevant factors that may be influencing its adoption. QAToCA delivers an assessment of how suitable conditions "and thus the likelihood for CA adoption" are. Results show that the high CA adoption potentials exhibited by the Malawi and Zambia case relate mostly to positive institutional factors. On the other hand, the low adoption potential of the Zimbabwe case, in spite of observed higher estimates, is attributed mainly to unstable and less secured market conditions for CA. In the case of Southern Burkina Faso, the potential for CA adoption is determined to be high, and this assessment deviates from lower observed figures. This is attributed mainly to strong competition of CA and livestock for residues in this region. Lastly, the high adoption potential found in Northern Burkina Faso is explained mainly by the fact that farmers here have no alternative other than to adopt the locally adapted CA system-Zaï farming. Results of this assessment should help promoters of CA in the given regions to reflect on their activities and to eventually adjust or redesign them based on a more explicit understanding of where problems and opportunities are found.

Plasmodium falciparum clearance rates in response to artesunate in Malian children with malaria: effect of acquired immunity.

BACKGROUND: Artemisinin resistance, a long parasite clearance half-life in response to artemisinin, has been described in patients with Plasmodium falciparum malaria in southeast Asia. Few baseline half-lives have been reported from Africa, where artemisinins were recently introduced. METHODS: We treated P. falciparum malaria in 215 Malian children aged 0.5-15 years with artesunate (0, 24, 48 hours) and amodiaquine (72, 96, 120 hours). We estimated half-life by measuring parasite density every 6 hours until undetectable and evaluated the effects of age, sex, ethnicity, and red blood cell (RBC) polymorphisms on half-life. We quantified the proportion of parasitized RBCs recognized by autologous immunoglobulin G (IgG). RESULTS: The geometric mean half-life was 1.9 hours (95% confidence interval, 1.8-2.0) and did not correlate with parasite ex vivo susceptibility to artemisinins. In a linear model accounting for host factors, half-life decreased by 4.1 minutes for every 1-year increase in age. The proportion of parasitized RBCs recognized by IgG correlated inversely with half-life (r = -0.475; P = .0006). CONCLUSIONS: Parasite clearance in response to artesunate is faster in Mali than in southeast Asia. IgG responses to parasitized RBCs shorten half-life and may influence this parameter in areas where age is not an adequate surrogate of immunity and correlates of parasite-clearing immunity have not been identified. CLINICAL TRIALS REGISTRATION: NCT00669084.

Nanoparticle tracking analysis of natural hemozoin from Plasmodium parasites.

BACKGROUND: Hemozoin is a byproduct of hemoglobin digestion crucial for parasite survival. It forms crystals that can be of interest as drug targets or biomarkers of malaria infection. However, hemozoin has long been considered as an amorphous crystal of simple morphology. Studying the consequences of biomineralization of this crystal during the parasite growth may provide more comprehensive evidence of its role during malaria. OBJECTIVES: This study aimed to investigate the interest of nanoparticles tracker analysis for measuring the concentration and size of hemozoin particles produced from different parasite sources and conditions. METHODS: Hemozoin was extracted from several clones of Plasmodium falciparum both asexual and sexual parasites. Hemozoin was also extracted from blood samples of malaria patients and from saliva of asymptomatic malaria carriers. Nanoparticles tracking analysis (NTA) was performed to assess the size and concentration of hemozoin. RESULTS: NTA data showed variation in hemozoin concentration, size, and crystal clusters between parasite clones, species, and stages. Among parasite clones, hemozoin concentration ranged from 131 to 2663 particles/infected red blood cell (iRBC) and size ranged from 149.6 ± 6.3 nm to 234.8 ± 40.1 nm. The mean size was lower for Plasmodium vivax (176 ± 79.2 nm) than for Plasmodium falciparum (254.8 ± 74.0 nm). Sexual NF54 parasites showed a 7.5-fold higher concentration of hemozoin particles (28.7 particles/iRBC) compared to asexual parasites (3.8 particles/iRBC). In addition, the mean hemozoin size also increased by approximately 60 % for sexual parasites. Compared to in vitro cultures of parasites, blood samples showed low hemozoin concentrations. CONCLUSIONS: This study highlights the potential of NTA as a useful method for analyzing hemozoin, demonstrating its ability to provide detailed information on hemozoin characterization. However, further research is needed to adapt the NTA for hemozoin analysis.

Gene expression analyses reveal differences in children's response to malaria according to their age.

In Bandiagara, Mali, children experience on average two clinical malaria episodes per year. However, even in the same transmission area, the number of uncomplicated symptomatic infections, and their parasitemia, can vary dramatically among children. We simultaneously characterize host and parasite gene expression profiles from 136 Malian children with symptomatic falciparum malaria and examine differences in the relative proportion of immune cells and parasite stages, as well as in gene expression, associated with infection and or patient characteristics. Parasitemia explains much of the variation in host and parasite gene expression, and infections with higher parasitemia display proportionally more neutrophils and fewer T cells, suggesting parasitemia-dependent neutrophil recruitment and/or T cell extravasation to secondary lymphoid organs. The child's age also strongly correlates with variations in gene expression: Plasmodium falciparum genes associated with age suggest that older children carry more male gametocytes, while variations in host gene expression indicate a stronger innate response in younger children and stronger adaptive response in older children. These analyses highlight the variability in host responses and parasite regulation during P. falciparum symptomatic infections and emphasize the importance of considering the children's age when studying and treating malaria infections.

Spatio-temporal analysis of malaria within a transmission season in Bandiagara, Mali.

BACKGROUND: Heterogeneous patterns of malaria transmission are thought to be driven by factors including host genetics, distance to mosquito breeding sites, housing construction, and socio-behavioural characteristics. Evaluation of local transmission epidemiology to characterize malaria risk is essential for planning malaria control and elimination programmes. The use of geographical information systems (GIS) techniques has been a major asset to this approach. To assess time and space distribution of malaria disease in Bandiagara, Mali, within a transmission season, data were used from an ongoing malaria incidence study that enrolled 300 participants aged under six years old". METHODS: Children's households were georeferenced using a handheld global position system. Clinical malaria was defined as a positive blood slide for Plasmodium falciparum asexual stages associated with at least one of the following signs: headache, body aches, fever, chills and weakness. Daily rainfall was measured at the local weather station.Landscape features of Bandiagara were obtained from satellite images and field survey. QGIS™ software was used to map malaria cases, affected and non-affected children, and the number of malaria episodes per child in each block of Bandiagara. Clusters of high or low risk were identified under SaTScan(®) software according to a Bernoulli model. RESULTS: From June 2009 to May 2010, 296 clinical malaria cases were recorded. Though clearly temporally related to the rains, Plasmodium falciparum occurrence persisted late in the dry season. Two "hot spots" of malaria transmission also found, notably along the Yamé River, characterized by higher than expected numbers of malaria cases, and high numbers of clinical episodes per child. Conversely, the north-eastern sector of the town had fewer cases despite its proximity to a large body of standing water which was mosquito habitat. CONCLUSION: These results confirm the existence of a marked spatial heterogeneity of malaria transmission in Bandiagara, providing support for implementation of targeted interventions.

Malian children infected with Plasmodium ovale and Plasmodium falciparum display very similar gene expression profiles.

Plasmodium parasites caused 241 million cases of malaria and over 600,000 deaths in 2020. Both P. falciparum and P. ovale are endemic to Mali and cause clinical malaria, with P. falciparum infections typically being more severe. Here, we sequenced RNA from nine pediatric blood samples collected during infections with either P. falciparum or P. ovale, and characterized the host and parasite gene expression profiles. We found that human gene expression varies more between individuals than according to the parasite species causing the infection, while parasite gene expression profiles cluster by species. Additionally, we characterized DNA polymorphisms of the parasites directly from the RNA-seq reads and found comparable levels of genetic diversity in both species, despite dramatic differences in prevalence. Our results provide unique insights into host-pathogen interactions during malaria infections and their variations according to the infecting Plasmodium species, which will be critical to develop better elimination strategies against all human Plasmodium parasites.

Gene expression analyses reveal differences in children's response to malaria according to their age.

In Bandiagara, Mali, children experience on average two clinical malaria episodes per season. However, even in the same transmission area, the number of uncomplicated symptomatic infections, and their parasitemia, vary dramatically among children. To examine the factors contributing to these variations, we simultaneously characterized the host and parasite gene expression profiles from 136 children with symptomatic falciparum malaria and analyzed the expression of 9,205 human and 2,484 Plasmodium genes. We used gene expression deconvolution to estimate the relative proportion of immune cells and parasite stages in each sample and to adjust the differential gene expression analyses. Parasitemia explained much of the variation in both host and parasite gene expression and revealed that infections with higher parasitemia had more neutrophils and fewer T cells, suggesting parasitemia-dependent neutrophil recruitment and/or T cell extravasation to secondary lymphoid organs. The child's age was also strongly correlated with gene expression variations. Plasmodium falciparum genes associated with age suggested that older children carried more male gametocytes, while host genes associated with age indicated a stronger innate response (through TLR and NLR signaling) in younger children and stronger adaptive immunity (through TCR and BCR signaling) in older children. These analyses highlight the variability in host responses and parasite regulation during P. falciparum symptomatic infections and emphasize the importance of considering the children's age when studying and treating malaria infections.