Parasite Populations Research Articles

Plasmodium falciparum histidine rich protein 2 (pfhrp2): an additional genetic marker suitable for anti-malarial drug efficacy trials

BackgroundGenotyping of the three Plasmodium falciparum polymorphic genes, msp1, msp2 and glurp, has been adopted as a standard strategy to distinguish recrudescence from new infection in drug efficacy clinical trials. However, the suitability of a particular gene is compromised in areas where its allelic variants distribution is significantly skewed, a phenomenon that might occur in isolated parasite populations or in areas of very low transmission. Moreover, observation of amplification bias has diminished the value of glurp as a marker.MethodsThe suitability of the polymorphic P. falciparum histidine-rich protein 2 (pfhrp2) gene was assessed to serve as an alternative marker using a PCR-sequencing or a PCR–RFLP protocol for genotyping of samples in drug efficacy clinical trials. The value of pfhrp2 was validated by side-by-side analyses of 5 admission-recrudescence sample pairs from Yemeni malaria patients.ResultsThe outcome of the single pfhrp2 gene discrimination analysis has been found consistent with msp1, msp2 and glurp pool genotyping analysis for the differentiation of recrudescence from new infection.ConclusionThe findings suggest that under the appropriate circumstances, pfhrp2 can serve as an additional molecular marker for monitoring anti-malarials efficacy. However, its use is restricted to endemic areas where only a minority of P. falciparum parasites lack the pfhrp2 gene.

Genetic diversity of Trypanosoma cruzi strains isolated from chronic chagasic patients and non-human hosts in the state of São Paulo, Brazil.

Trypanosoma cruzi shows an exuberant genetic diversity. Currently, seven phylogenetic lineages, called discrete typing units (DTUs), are recognised: TcI-TcVI and Tcbat. Despite advances in studies on T. cruzi and its populations, there is no consensus regarding its heterogeneity. This study aimed to perform molecular characterisation of T. cruzi strains, isolated in the state of São Paulo, to identify the DTUs involved and evaluate their genetic diversity. T. cruzi strains were isolated from biological samples of chronic chagasic patients, marsupials and triatomines through culture techniques and subjected to molecular characterisation using the fluorescent fragment length barcoding (FFLB) technique. Subsequently, the results were correlated with complementary information to enable better discrimination between the identified DTUs. It was possible to identify TcI in two humans and two triatomines; TcII/VI in 19 humans, two marsupials and one triatomine; and TcIII in one human host, an individual that also presented a result for TcI, which indicated the possibility of a mixed infection. Regarding the strains characterised by the TcII/VI profile, the correlation with complementary information allowed to suggest that, in general, these parasite populations indeed correspond to the TcII genotype. The TcII/VI profile, associated with domestic cycles and patients with chronic Chagas disease, was the most prevalent among the identified DTUs. Furthermore, the correlation of the study results with complementary information made it possible to suggest that TcII is the predominant lineage of this work.

Resetting var Gene Transcription in Plasmodium falciparum.

One of the key mechanisms contributing to the virulence of Plasmodium falciparum is its ability to undergo antigenic switching among antigenically distinct variants of the PfEMP1 adhesive proteins, encoded by the var gene family. To avoid premature exposure of its antigenic repertoire, the parasite transcribes its var genes in a mutually exclusive manner, and switch expression at a very slow rate. This process is epigenetically regulated and it relies on "epigenetic memory," which imprints the single active var gene to remain active for multiple replication cycles. Erasing this epigenetic memory in parasites grown in culture resembles parasites, which egress from the liver. It could therefore be of interest for investigating var switching patterns at the onset of malaria infections. In addition, this procedure could be used for creating heterogeneity of var expression among parasite populations. The methodology described here for resetting of var gene expression is based on promoter titration, also known as molecular sponging.

Single-Cell Sorting of Plasmodium falciparum-Infected Erythrocytes Expressing Particular PfEMP1 Variants.

Cultures of Plasmodium falciparum often contain a heterogeneous parasite population. However, several studies require analysis of single infected erythrocytes (IEs) or a clonal parasite population derived from a single parasite. This protocol describes an efficient method for cloning by using fluorescence-activated cell sorting (FACS). For this, an antibody for a particular IEs surface protein it is added to the cell mixture to separate positive and negative IEs for that marker. After the separation, the viable homogeneous population can be used to grow in culture or for molecular analysis.

CRISPR-Cas9 Editing of the Plasmodium falciparum Genome: Special Applications.

The virulence of Plasmodium falciparum has been attributed in large part to the expression on the surface of infected red blood cells of the variant surface antigen Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1). Different forms of this protein are encoded by individual members of the multicopy gene family called var. Two attributes of the var gene family are key to the pathogenesis of malaria caused by P. falciparum; the hyperrecombinogenic nature of the var gene family that continuously generates antigenic diversity within parasite populations, and the ability of parasites to express only a single var gene at a time and to switch which gene is expressed over the course of an infection. The unique attributes of CRISPR-Cas9 have been applied to help decipher the molecular mechanisms underlying these unusual properties of the var gene family, both as a source of the DNA double strand breaks that initiate var gene recombination and as a way to recruit molecular probes to specific regions of the genome. In this chapter, we describe these somewhat unusual applications of the CRISPR-Cas9 system.

The importance of biological vision in the control of plant pests

Animal populations living on one or more plants, as well as the parasitic and predatory populations built around them, and those living from the carcass, waste, and other populations of all these plants constitute a life-changer held together by specific laws. It is therefore essential that the ratio between plant protection products, on the one hand, and entomological ecological research, on the other hand, should very soon change. Only agrocoenologists are capable to carry out the research task, which is very closely related to plant protection already that examines the immediate and more distant effects of the broad variety of protection methods, particularly those by chemical control. We allude to the agronomist, familiar with biology and not changing his farmland exceedingly often; who can gradually, year after year, compile the building blocks of experience; who can keep an eye on the major alterations in wildlife upon the anthropogenic activities that transform nature; who can record changes in the bulk of pests, their disappearance and reemergence; and who could observe the impacts of plant protection work with a critical eye. We must strive to find processes based on biological-ecological research, practically pest by pest, that allow the greatest use of natural limiting factors by restraining chemical treatments to the narrowest and most appropriate schedule. In our article, we describe some methods and principles of the implementation of a biological approach and ecological plant protection.

Costly defense traits in structured populations

We propose a model for the dynamics of frequencies of a costly defense trait. More precisely, we consider Lotka-Volterra-type models involving a prey (or host) population consisting of two types and a predator (or parasite) population, where one type of prey individuals - modeling carriers of a defense trait - is more effective in defending against the predators but has a weak reproductive disadvantage. Under certain assumptions we prove that the relative frequency of these defenders in the total prey population converges to spatially structured Wright-Fisher diffusions with frequency-dependent migration rates. For the many-demes limit (mean-field approximation) hereof, we show that the defense trait goes to fixation/extinction if and only if the selective disadvantage is smaller/larger than an explicit function of the ecological model parameters.

Temporal effects and changes in the parasitic community ofProchilodus lineatus(Valenciennes, 1837) (Characiformes: Prochilodontidae) in a floodplain

The construction of dams causes several impacts on aquatic environments, altering the flow of rivers, environmental variables, and all biota present, including parasites. Little is known about how the parasitic community can be influenced in the long term by environmental changes. In this study, it was expected that the impacts caused by environmental disturbances will be directly reflected by the composition of the parasite populations. We evaluated the change in the structure of the Prochilodus lineatus endoparasite community between two periods sampled 15 years apart in the upper Paraná River floodplain. There was a significant difference in the weight-length relationship of P. lineatus between these periods and a total of 15 species of parasites were found: 11 species in Period 1 and nine species in Period 2 and five species occurred in both periods. The species richness and diversity were higher in Period 1, and we observed that the correlation of descriptors (richness, diversity and evenness) increased with fish length in this period. In both periods, digeneans numerically dominated the parasitic community, and we verified changes in the composition of parasites between periods. Both the host and the parasites were possibly affected by the environmental impacts resulting from the construction of dams over time, and it is noteworthy that complex life cycle parasites such as Digenea and Acanthocephala require intermediate hosts to complete their life cycle, and the population responds to fluctuations in the face of modified environments.

ThePlasmodium falciparuminfection status of two major anopheline vectors in three hyper-, meso-, and hypoendemic districts in Odisha, India

AbstractIn-depth understanding of malaria transmission dynamics in a region can be assessed by identifying the vector populations of infected mosquitoes,Anophelesspp. (Diptera: Culicidae), and by quantifying the infectiousness extent. In this study of malaria transmission dynamics relating to vector incrimination in three districts of Odisha, India – hyperendemic Kalahandi, mesoendemic Bargarh, and hypoendemic Cuttack – we examined how quality and quantity of plasmodial infection rates vary among mosquito species and their organs and among districts. The minimum infection rate ofPlasmodium falciparumsporozoite forAnopheles culicifacieswas highest in Kalahandi and nil in Cuttack. However, forA. annularis, the rate was highest in Cuttack, followed by Kalahandi and Bargarh. In Kalahandi, the gland-positive rate was higher forA. culicifacies, but in Cuttack, it was higher forA. annularis. To quantify plasmodium infection in salivary glands and guts of anopheline vectors, quantitative polymerase chain reaction was performed. We observed higher parasite density in glands than in guts of vector mosquitoes in the three districts. The findings demonstrate that plasmodium-infected vectors preferentially bind sporozoites to salivary glands in the three study areas. The results improve understanding of infection status within malaria vectors and how parasite population density may affect transmissibility, which will, in turn, provide baseline evidence to develop intervention measures.

Genetic Diversity of Schistosoma haematobium in Sub-Saharan Africa: A Systematic Review

Urogenital schistosomiasis caused by the parasite Schistosoma haematobium is the most common form of schistosomiasis. This parasite has a high potential for genetic exchange within parasite populations giving rise to the genetic diversity that is important for its survival. Genetic differences may lead to some parasite strains being more immunogenic which may have a negative impact on management and control of schistosomiasis. Therefore, understanding these genetic differences in the parasite may lead to better management of the disease. The aim of this review was to systematically review scientific literature on the genetic diversity and population structuring of S. haematobium and the methods used across sub-Saharan African countries. A literature search was done on PubMed, African Journals online and Google scholar using predefined search terms such as urinary schistosomiasis, S. haematobium, genetic diversity in sub-Saharan Africa in combination with Boolean operators (AND, OR). The search included studies published from 2000-2020 that emphasised on genetic diversity of S. haematobium in sub-Saharan Africa. A country in sub-Saharan Africa was included if had a study that determined the genetic diversity of S. haematobium Sixteen study articles from 18 sub-Saharan African countries met the inclusion criteria The genetic diversity of S. haematobium varied from low to high using different methods. Most studies conducted in these countries showed a high genetic diversity of S. haematobium 18/36 (50%) studies. Four methods namely, restriction fragment length polymorphism, randomly amplified polymorphic DNA, DNA barcoding and Microsatellite markers were used to determine diversity In these studies, measures of genetic diversity such as number of alleles per minute or allelic richness, heterozygosity, number of genotypes and unique haplotypes were used. Microsatellites were the most commonly used method and the studies reported number of alleles per locus ranging from 2-19 alleles per locus and heterozygosity of 6 to 71% in some studies. . The highest number of studies were conducted in West Africa Nigeria and Zimbabwe , 4/36(11%) each. Results show the need for continued monitoring of genetic variations in S. haematobium in sub- Saharan Africa. This will aid in understanding the epidemiology of disease, advancing novel treatment and vaccine strategies.

Come with me if you want to live: sympatric parasites follow different transmission routes through aquatic host communities

Community composition, including the relative density of each host species, plays a vital role in the transmission of parasites or disease in freshwater ecosystems. Whereas some host species can effectively transmit parasites, others can act as dead ends (non-viable transmission routes), accumulating large numbers of parasites throughout their life, thus becoming important sinks for parasite populations. Although population sinks have been identified in certain host-parasite systems, robust field estimates of the proportions of parasites that are lost to these hosts are lacking. Here, we quantified the distribution of encysted larval hairworms (phylum Nematomorpha), common parasites in lotic ecosystems, in two subalpine stream communities of New Zealand. With parasite and host population densities calculated per m2, we identified which host species most likely contributed to the transmission of three sympatric hairworm morphotypes identified in both streams, and which species acted as population sinks. We also tested for seasonal patterns and peaks in the abundance of each morphotype in the two communities over the sampling season. Finally, we tested whether hosts emerging from the streams had comparable abundances of hairworm morphotypes throughout the sampling period. For each morphotype, different key sets of host species harboured more hairworms on average (abundance) than others, depending on the stream. For one morphotype in particular, two species of hosts were found to be important population sinks that inhibited over a third of these parasites from completing their life cycle. We also observed a clear peak in abundance for another hairworm morphotype during summer. Our data suggest that hosts emerging from the streams matched their aquatic counterparts with respect to hairworm abundance, indicating no infection-dependent reduction in emergence success. Our findings suggest that, depending on relative community composition, sympatric parasites follow different host transmission pathways, some of which lead to dead ends that potentially impact overall infection dynamics. In turn, this information can help us understand the spread or emergence of disease in both freshwater and terrestrial environments, since hairworms infect terrestrial arthropods to complete their life cycle.

Distinguishing gene flow between malaria parasite populations.

Measuring gene flow between malaria parasite populations in different geographic locations can provide strategic information for malaria control interventions. Multiple important questions pertaining to the design of such studies remain unanswered, limiting efforts to operationalize genomic surveillance tools for routine public health use. This report examines the use of population-level summaries of genetic divergence (FST) and relatedness (identity-by-descent) to distinguish levels of gene flow between malaria populations, focused on field-relevant questions about data size, sampling, and interpretability of observations from genomic surveillance studies. To do this, we use P. falciparum whole genome sequence data and simulated sequence data approximating malaria populations evolving under different current and historical epidemiological conditions. We employ mobile-phone associated mobility data to estimate parasite migration rates over different spatial scales and use this to inform our analysis. This analysis underscores the complementary nature of divergence- and relatedness-based metrics for distinguishing gene flow over different temporal and spatial scales and characterizes the data requirements for using these metrics in different contexts. Our results have implications for the design and implementation of malaria genomic surveillance studies.

Scanning electron microscopy of Quilonia renniei from Asian elephants revealing variation in coronal leaflet number.

Although parasitic nematodes in the genera Murshidia and Quilonia (family Strongylidae) are recognized as major gastrointestinal parasites in Asian elephants, they have been poorly studied. Recently, light micrographs of these parasites in Myanmar have been presented, almost 100 years after the original drawings. However, the number of coronal leaflets, a key taxonomic feature of Quilonia species, has not been precisely determined based on light microscopy. The current study aimed to determine the exact number of coronal leaflets in Quilonia renniei specimens from Asian elephants in Myanmar. On the basis of scanning electron micrographs, leaflet number in females (19–20, average 19.7, n = 9) was significantly higher (P < 0.005) than that in males (16–19, average 18.1, n = 8). This compares with 18 coronal leaflets indicated in the original species description. Specimens bearing 19 coronal leaflets were most numerous, followed by those with 20 leaflets. Median-joining network analysis of mitochondrial cytochrome c oxidase subunit I gene sequences with 16 haplotypes from 19 individuals revealed no clear association between parasite populations and the number of coronal leaflets. These results highlight the importance of determining the number of coronal leaflets in the taxonomy of Q. renniei and other related Quilonia species infecting Asian elephants.

Genome instability drives epistatic adaptation in the human pathogen Leishmania

How genome instability is harnessed for fitness gain despite its potential deleterious effects is largely elusive. An ideal system to address this important open question is provided by the protozoan pathogen Leishmania, which exploits frequent variations in chromosome and gene copy number to regulate expression levels. Using ecological genomics and experimental evolution approaches, we provide evidence that Leishmania adaptation relies on epistatic interactions between functionally associated gene copy number variations in pathways driving fitness gain in a given environment. We further uncover posttranscriptional regulation as a key mechanism that compensates for deleterious gene dosage effects and provides phenotypic robustness to genetically heterogenous parasite populations. Finally, we correlate dynamic variations in small nucleolar RNA (snoRNA) gene dosage with changes in ribosomal RNA 2'-O-methylation and pseudouridylation, suggesting translational control as an additional layer of parasite adaptation. Leishmania genome instability is thus harnessed for fitness gain by genome-dependent variations in gene expression and genome-independent compensatory mechanisms. This allows for polyclonal adaptation and maintenance of genetic heterogeneity despite strong selective pressure. The epistatic adaptation described here needs to be considered in Leishmania epidemiology and biomarker discovery and may be relevant to other fast-evolving eukaryotic cells that exploit genome instability for adaptation, such as fungal pathogens or cancer.

Malaria protection due to sickle haemoglobin depends on parasite genotype

Host genetic factors can confer resistance against malaria1, raising the question of whether this has led to evolutionary adaptation of parasite populations. Here we searched for association between candidate host and parasite genetic variants in 3,346 Gambian and Kenyan children with severe malaria caused by Plasmodium falciparum. We identified a strong association between sickle haemoglobin (HbS) in the host and three regions of the parasite genome, which is not explained by population structure or other covariates, and which is replicated in additional samples. The HbS-associated alleles include nonsynonymous variants in the gene for the acyl-CoA synthetase family member2–4PfACS8 on chromosome 2, in a second region of chromosome 2, and in a region containing structural variation on chromosome 11. The alleles are in strong linkage disequilibrium and have frequencies that covary with the frequency of HbS across populations, in particular being much more common in Africa than other parts of the world. The estimated protective effect of HbS against severe malaria, as determined by comparison of cases with population controls, varies greatly according to the parasite genotype at these three loci. These findings open up a new avenue of enquiry into the biological and epidemiological significance of the HbS-associated polymorphisms in the parasite genome and the evolutionary forces that have led to their high frequency and strong linkage disequilibrium in African P. falciparum populations.

Characterization of the mitochondrial genome of Tetrameres grusi and insights into the phylogeny of Spirurina.

Tetrameres grusi is a significant parasitic nematode of cranes that is classified into suborder Spirurina. However, for more than a century, this classification has been controversial. Mitochondrial genomes are valuable resources for parasite taxonomy, population genetics and systematics studies. Here, the mitochondrial genome of T. grusi was determined and subsequently compared with those from Spirurina species using concatenated datasets of amino acid sequences predicted from mitochondrial protein-coding genes. The complete mitochondrial genome of T. grusi is circular with 13,709 bp, and it contains 12 protein-coding genes, 22 transfer RNA genes, two ribosomal RNA genes and one non-coding region. All of the protein-coding genes are transcribed in the same direction. There were 18 intergenic spacers of 1–44 bp, and six locations with gene overlaps, ranging from 1 bp to 28 bp, in the mitochondrial genome of T. grusi. The AT content of this mitochondrial genome was 71.56%. This was similar to mitochondrial genomes of other Spirurina species, which also exhibited strong AT content bias, not only in the nucleotide composition but also in codon usage. The sequenced mitogenomes of the 25 Spirurina nematodes showed three classes of gene arrangements based on the 12 protein-coding genes, and the gene arrangement of the T. grusi mitochondrial genome belonged to the Class I. Phylogenetic analyses using mitochondrial genomes of 25 Spirurina nematodes revealed that T. grusi (Habronematoidea) was closer to Gongylonema pulchrum (Spiruroidea) than Spirocerca lupi (Thelazioidea). The availability of the complete mitochondrial genome sequence of T. grusi provides new and useful genetic markers for further studies on Spirurina nematodes.

Biosolarization restructures soil bacterial communities and decreases parasitic nematode populations

Biosolarization is a soil disinfestation technology that combines passive solar heating and organic amendments to generate multiple pest-inactivating stressors. The objectives of this study were to assess the performance of almond (Prunus dulcis) processing residues in biosolarization to control infestations of root lesion (Pratylenchus vulnus) and ring nematodes (Mesocriconema xenoplax) during the pre-planting period of an almond orchard. The broader effects on soil microbial community composition were assessed by 16S rRNA gene sequencing immediately before and after biosolarization, as well as two months after treatment ended. Soil organic acids with nematicidal activity peaked after 9 days in biosolarized plots. Temperatures in biosolarized plots and solarized control plots reached maximum values 9–12 °C higher than untreated control plots. Biosolarization and solarization also enriched for specific Firmicutes, Actinobacteria, and Proteobacteria taxa associated with nematode suppression, some of which remained enriched two months post treatment. Biosolarization led to 100% mortality of P. vulnus in the upper 0–30 cm soil layer by 9 days of treatment. In contrast, reductions in plant-parasitic nematode densities between non-amended, solarized treatments and untreated controls were not observed until day 41 of treatment.

Genetic polymorphism of Trypanosoma cruzi bloodstream populations in adult chronic indeterminate Chagas disease patients from the E1224 clinical trial.

The role that the genetic diversity of natural Trypanosoma cruzi populations plays in response to trypanocidal treatment of chronic Chagas disease (CD) patients remains to be understood. We analysed the genetic polymorphisms of parasite bloodstream populations infecting chronic CD patients enrolled in the E1224 clinical trial. A total of 506 baseline and post-treatment follow-up samples from 188 patients were analysed. T. cruzi satellite DNA (satDNA) was amplified and sequenced using cruzi1/cruzi2 primers, and samples with TcI/III, TcII, TcIV or hybrid satDNA sequences were identified. Minicircle signatures were obtained after kinetoplast DNA amplification using 121/122 primers and restriction enzyme digestion. Genetic distances between baseline and post-treatment minicircle signatures were estimated using the Jaccard coefficient. At baseline, 74.3% TcII, 17.9% hybrid and 7.8% TcI/III satDNA sequences were found, whereas at the end of follow-up the distribution was 55.2% TcII, 35.2% hybrid and 9.5% TcI/III. The placebo arm was the treatment group with the highest variation of satDNA sequences between baseline and post-treatment follow-up. Genetic distances between baseline and post-treatment minicircle signatures were similar among all treatment arms. No association between minicircle signature variability and satDNA type distribution was found. Genetic variability of T. cruzi bloodstream populations during post-treatment follow-up did not differ from that observed during chronic infection in the absence of treatment, suggesting that there were no selection events of E1224-resistant parasite populations. This is the first report documenting the genetic polymorphism of natural T. cruzi populations in chronic patients in the context of clinical trials with trypanocidal drugs.

Anthelmintic Resistance and Its Mechanism: A Review.

Helminths are a various types of parasites causing a major health problem for animals in different parts of the globe. Control of helminthiasis has largely relied on the use of pharmaceutical anthelmintics. Unfortunately, the exhaustive use of anthelmintic drugs has led to a serious and dramatic level of anthelmintic resistance. Anthelmintic resistance is a heritable loss of sensitivity of an anthelmintic in a parasite population that was in the past susceptible to the same anthelmintic. The development of anthelmintic resistance is evident to different helminths of almost every animal species and to different groups of anthelmintic in several continents. Frequent treatment, underdosing, genetics of the parasite, and targeting and timing of mass treatment are predisposing factors for anthelmintic resistance. Upregulation of cellular efflux mechanisms, an increase in drug metabolism, a change in drug receptor sites that reduces drug binding or the functional consequences of drug binding, and a decrease in drug receptor abundance through reduced expression within the parasite are the main mechanisms of anthelmintic resistance. In vivo method like fecal egg count reduction test and in vitro method such as egg hatch assays, larval motility test, larval development test and PCR can be used for the detection of anthelmintic resistance. Proper utilization of anthelmintic drugs, using combined anthelmintic and applying other alternatives are essential strategies to slow down the development of anthelmintic resistance. As anthelmintic resistance is a serious challenge throughout the world, proper utilization of the existing anthelmintics and reducing dependence on anthelmintics should be implemented to reduce its challenge.

Spatiotemporal Dynamic of the RTS,S/AS01 Malaria Vaccine Target Antigens in Senegal.

The RTS,S/AS01 malaria vaccine confers only moderate protection against malaria. Evidence suggests that the effectiveness of the RTS,S/AS01 vaccine depends upon the parasite population genetics, specifically regarding the circumsporozoite protein haplotypes in the population. We investigated Plasmodium falciparum circumsporozoite protein (PfCSP) gene sequences from two endemic sites in 2018 in Senegal. The PfCSP sequences were compared with those retrieved from the Pf3k genome database. In the central repeat region of PfCSP, the distribution of haplotypes differed significantly between the two study sites (Fisher's exact test, P < 0.001). No 3D7 vaccine strain haplotype was observed in this locus. In the C-terminal region, there was no significant difference in haplotypes distribution between Kedougou and Diourbel (Fischer's exact test, P = 0.122). The 3D7 haplotype frequency was 8.4% in early samples (2001-2011), but then it contracted in the subsequent years. The extensive plasticity of the P. falciparum genes coding the RTS,S/AS01 vaccine target antigens may influence the immune responses to circulating alleles. Monitoring the genetic diversity baseline and its dynamics over time and space would be instrumental in rationally improving the malaria RTS,S/AS01 vaccine and/or its implementation schedule.