Informing and evaluating malaria control efforts relies on knowledge of local transmission dynamics. Serological and molecular tools have demonstrated great sensitivity to quantify transmission intensity in low endemic settings where the sensitivity of traditional methods is limited. Filter paper blood spots are commonly used a source of both DNA and antibodies. To enhance the operational practicability of malaria surveys, a method is presented for combined DNA extraction and antibody elution.

In order to harmonize results for the detection and quantification of Plasmodium falciparum DNA by nucleic acid amplification technique (NAT)-based assays, a World Health Organization (WHO) collaborative study was performed, evaluating a series of candidate standard preparations.

The genomic diversity of Plasmodium malariae malaria parasites is understudied, partly because infected individuals tend to present with low parasite densities, leading to difficulties in obtaining sufficient parasite DNA for genome analysis. Selective whole genome amplification (SWGA) increases the relative levels of pathogen DNA in a clinical sample, but has not been adapted for P. malariae parasites. Here we design customized SWGA primers which successfully amplify P. malariae DNA extracted directly from unprocessed clinical blood samples obtained from patients with P. malariae-mono-infections from six countries, and further test the efficacy of SWGA on mixed infections with other Plasmodium spp. SWGA enables the successful whole genome sequencing of samples with low parasite density (i.e. one sample with a parasitaemia of 0.0064% resulted in 44% of the genome covered by ≥ 5 reads), leading to an average 14-fold increase in genome coverage when compared to unamplified samples. We identify a total of 868,476 genome-wide SNPs, of which 194,709 are unique across 18 high-quality isolates. After exclusion of the hypervariable subtelomeric regions, a high-quality core subset of 29,899 unique SNPs is defined. Population genetic analysis suggests that P. malariae parasites display clear geographical separation by continent. Further, SWGA successfully amplifies genetic regions of interest such as orthologs of P. falciparum drug resistance-associated loci (Pfdhfr, Pfdhps, Pfcrt, Pfk13 and Pfmdr1), and several non-synonymous SNPs were detected in these genes. In conclusion, we have established a robust SWGA approach that can assist whole genome sequencing of P. malariae, and thereby facilitate the implementation of much-needed large-scale multi-population genomic studies of this neglected malaria parasite. As demonstrated in other Plasmodia, such genetic diversity studies can provide insights into the biology underlying the disease and inform malaria surveillance and control measures.

MalariaGEN is a data-sharing network that enables groups around the world to work together on the genomic epidemiology of malaria. Here we describe a new release of curated genome variation data on 7,000 Plasmodium falciparum samples from MalariaGEN partner studies in 28 malaria-endemic countries. High-quality genotype calls on 3 million single nucleotide polymorphisms (SNPs) and short indels were produced using a standardised analysis pipeline. Copy number variants associated with drug resistance and structural variants that cause failure of rapid diagnostic tests were also analysed.  Almost all samples showed genetic evidence of resistance to at least one antimalarial drug, and some samples from Southeast Asia carried markers of resistance to six commonly-used drugs. Genes expressed during the mosquito stage of the parasite life-cycle are prominent among loci that show strong geographic differentiation. By continuing to enlarge this open data resource we aim to facilitate research into the evolutionary processes affecting malaria control and to accelerate development of the surveillance toolkit required for malaria elimination.

Atovaquone-proguanil is one of the most commonly prescribed malaria prophylactic drugs. However, sporadic mutations conferring resistance to atovaquone have been detected in recent years associated with single nucleotide polymorphisms (SNPs) in the Plasmodium falciparum cytochrome b ( pfcytb) gene. Monitoring polymorphisms linked with resistance is essential in assessing the prevalence of drug resistance and may help in designing strategies for malaria control. Several approaches have been used to study genetic polymorphisms associated with antimalarial drug resistance. However, they either lack high throughput capacity or are expensive in time or money. Ligase detection reaction fluorescent microsphere assay (LDR-FMA) provides a high-throughput method to detect genetic polymorphisms in P. falciparum. In this study, we have created primers to detect SNPs associated with clinically relevant atovaquone resistance using LDR-FMA and validated them in clinical samples. Four SNPs from pfcytb gene were analyzed using LDR-FMA. The results were 100% consistent with DNA sequence data, indicating that this method has potential as a tool to detect genetic polymorphisms associated with atovaquone resistance in P. falciparum.

Parasite-encoded variant surface antigens (VSAs) like the var gene-encoded Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) family are responsible for antigenic variation and infected red blood cell (RBC) cytoadhesion in P. falciparum malaria. Parasites causing severe malaria in nonimmune patients tend to express a restricted subset of VSA (VSA(SM)) that differs from VSA associated with uncomplicated malaria and asymptomatic infection (VSA(UM)). We compared var gene transcription in unselected P. falciparum clone 3D7 expressing VSA(UM) to in vitro-selected sublines expressing VSA(SM) to identify PfEMP1 responsible for the VSA(SM) phenotype. Expression of VSA(SM) was accompanied by up-regulation of Group A var genes. The most prominently up-regulated Group A gene (PFD1235w/MAL7P1.1) was translated into a protein expressed on the infected RBC surface. The proteins encoded by Group A var genes, such as PFD1235w/MAL7P1.1, appear to be involved in the pathogenesis of severe disease and are thus attractive candidates for a vaccine against life-threatening P. falciparum malaria.

Seasonal Malaria Chemoprevention (SMC) with sulfadoxine-pyrimethamine (SP) plus amodiaquine (AQ), given each month during the transmission season, is recommended for children living in areas of the Sahel where malaria transmission is highly seasonal. The recommendation for SMC is currently limited to children under five years of age, but, in many areas of seasonal transmission, the burden in older children may justify extending this age limit. This study was done to determine the effectiveness of SMC in Senegalese children up to ten years of age.

Seasonal malaria chemoprevention (SMC) is recommended in the Sahel region of Africa for children under 5 years of age, for up to 4 months of the year. It may be appropriate to include older children, and to provide protection for more than 4 months. We evaluated the effectiveness of SMC using sulfadoxine-pyrimethamine plus amodiaquine given over 5 months to children under 10 years of age in Saraya district in south-east Senegal in 2011.

Deletions of the Plasmodium falciparum hrp2 and hrp3 genes can affect the performance of HRP2-based malaria rapid diagnostic tests (RDTs). Such deletions have been reported from South America, India and Eritrea. Whether these parasites are widespread in East Africa is unknown. A total of 274 samples from asymptomatic children in Mbita, western Kenya, and 61 genomic  data from Kilifi, eastern Kenya, were available for analysis. PCR-confirmed samples were investigated for the presence of pfhrp2 and pfhrp3 genes. In samples with evidence of deletion, parasite presence was confirmed by amplifying three independent genes. We failed to amplify pfhrp2 from 25 of 131 (19.1%) PCR-confirmed samples. Of these, only 8 (10%) samples were microscopic positive and were classified as pfhrp2-deleted. Eight microscopically-confirmed pfhrp2-deleted samples with intact pfhrp3 locus were positive by HRP2-based RDT. In addition, one PCR-confirmed infection showed a deletion at the pfhrp3 locus. One genomic sample lacked pfhrp2 and one lacked pfhrp3. No sample harbored parasites lacking both genes. Parasites lacking pfhrp2 are present in Kenya, but may be detectable by HRP-based RDT at higher parasitaemia, possibly due to the presence of intact pfhrp3. These findings warrant further systematic study to establish prevalence and diagnostic significance.

Circumsporozoite protein (CS) is a leading vaccine antigen for falciparum malaria, but is highly polymorphic in natural parasite populations. The factors driving this diversity are unclear, but non-random assortment of the T cell epitopes TH2 and TH3 has been observed in a Kenyan parasite population. The recent publication of the crystal structure of the variable C terminal region of the protein allows the assessment of the impact of diversity on protein structure and T cell epitope assortment. Using data from the Gambia (55 isolates) and Malawi (235 isolates), we evaluated the patterns of diversity within and between epitopes in these two distantly-separated populations. Only non-synonymous mutations were observed with the vast majority in both populations at similar frequencies suggesting strong selection on this region. A non-random pattern of T cell epitope assortment was seen in Malawi and in the Gambia, but structural analysis indicates no intramolecular spatial interactions. Using the information from these parasite populations, structural analysis reveals that polymorphic amino acids within TH2 and TH3 colocalize to one side of the protein, surround, but do not involve, the hydrophobic pocket in CS, and predominately involve charge switches. In addition, free energy analysis suggests residues forming and behind the novel pocket within CS are tightly constrained and well conserved in all alleles. In addition, free energy analysis shows polymorphic residues tend to be populated by energetically unfavorable amino acids. In combination, these findings suggest the diversity of T cell epitopes in CS may be primarily an evolutionary response to intermolecular interactions at the surface of the protein potentially counteracting antibody-mediated immune recognition or evolving host receptor diversity.

Malaria elimination strategies require surveillance of the parasite population for genetic changes that demand a public health response, such as new forms of drug resistance. Here we describe methods for the large-scale analysis of genetic variation in Plasmodium falciparum by deep sequencing of parasite DNA obtained from the blood of patients with malaria, either directly or after short-term culture. Analysis of 86,158 exonic single nucleotide polymorphisms that passed genotyping quality control in 227 samples from Africa, Asia and Oceania provides genome-wide estimates of allele frequency distribution, population structure and linkage disequilibrium. By comparing the genetic diversity of individual infections with that of the local parasite population, we derive a metric of within-host diversity that is related to the level of inbreeding in the population. An open-access web application has been established for the exploration of regional differences in allele frequency and of highly differentiated loci in the P. falciparum genome.

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