Malarial anaemia is characterized by destruction of malaria infected red blood cells and suppression of erythropoiesis. Interleukin 12 (IL12) significantly boosts erythropoietic responses in murine models of malarial anaemia and decreased IL12 levels are associated with severe malarial anaemia (SMA) in children. Based on the biological relevance of IL12 in malaria anaemia, the relationship between genetic polymorphisms of IL12 and its receptors and SMA was examined.

One of the most commonly used molecular test for malaria diagnosis is the polymerase chain reaction (PCR)-based amplification of the 18S ribosomal DNA (rDNA) gene. Published diagnostic assays based on the 18S gene include the "gold standard" nested assay, semi-nested multiplex assay, and one tube multiplex assay. To our knowledge, no one has reported whether the two multiplex methods are better at detecting mixed Plasmodium infections compared to the nested assay using known quantities of DNA in experimentally mixed cocktails.

With renewed interest in malaria elimination, island environments present unique opportunities to achieve this goal. However, as transmission decreases, monitoring and evaluation programmes need increasingly sensitive tools to assess Plasmodium falciparum and Plasmodium vivax exposure. In 2009, to assess the role of serological markers in evaluating malaria transmission, a cross-sectional seroprevalence study was carried out in Tanna and Aneityum, two of the southernmost islands of the Vanuatu archipelago, areas where malaria transmission has been variably reduced over the past few decades.

Recently, a real-time PCR assay known as photo-induced electron transfer (PET)-PCR which relies on self-quenching primers for the detection of Plasmodium spp. and Plasmodium falciparum was described. PET-PCR assay was found to be robust, and easier to use when compared to currently available real-time PCR methods. The potential of PET-PCR for molecular detection of malaria parasites in a nationwide malaria community survey in Haiti was investigated.

Previous work suggests that Brazilian Plasmodium falciparum has limited genetic diversity and a history of bottlenecks, multiple reintroductions due to human migration, and clonal expansions. We hypothesized that Brazilian P. falciparum would exhibit clonal structure. We examined isolates collected across two decades from Amapá, Rondônia, and Pará state (n = 190). By examining more microsatellites markers on more chromosomes than previous studies, we hoped to define the extent of low diversity, linkage disequilibrium, bottlenecks, population structure, and parasite migration within Brazil. We used retrospective genotyping of samples from the 1980s and 1990s to explore the population genetics of SP resistant dhfr and dhps alleles. We tested an existing hypothesis that the triple mutant dhfr mutations 50R/51I/108N and 51I/108N/164L developed in southern Amazon from a single origin of common or similar parasites. We found that Brazilian P. falciparum had limited genetic diversity and isolation by distance was rejected, which suggests it underwent bottlenecks followed by migration between sites. Unlike Peru, there appeared to be gene flow across the Brazilian Amazon basin. We were unable to divide parasite populations by clonal lineages and pairwise FST were common. Most parasite diversity was found within sites in the Brazilian Amazon, according to AMOVA. Our results challenge the hypothesis that triple mutant alleles arose from a single lineage in the Southern Amazon. SP resistance, at both the double and triple mutant stages, developed twice and potentially in different regions of the Brazilian Amazon. We would have required samples from before the 1980s to describe how SP resistance spread across the basin or describe the complex internal migration of Brazilian parasites after the colonization efforts of past decades. The Brazilian Amazon basin may have sufficient internal migration for drug resistance reported in any particular region to rapidly spread to other parts of basin under similar drug pressure.

Malaria in sub-Saharan Africa has historically been almost exclusively attributed to Plasmodium falciparum (Pf). Current diagnostic and surveillance systems in much of sub-Saharan Africa are not designed to identify or report non-Pf human malaria infections accurately, resulting in a dearth of routine epidemiological data about their significance. The high prevalence of Duffy negativity provided a rationale for excluding the possibility of Plasmodium vivax (Pv) transmission. However, review of varied evidence sources including traveller infections, community prevalence surveys, local clinical case reports, entomological and serological studies contradicts this viewpoint. Here, these data reports are weighted in a unified framework to reflect the strength of evidence of indigenous Pv transmission in terms of diagnostic specificity, size of individual reports and corroboration between evidence sources. Direct evidence was reported from 21 of the 47 malaria-endemic countries studied, while 42 countries were attributed with infections of visiting travellers. Overall, moderate to conclusive evidence of transmission was available from 18 countries, distributed across all parts of the continent. Approximately 86.6 million Duffy positive hosts were at risk of infection in Africa in 2015. Analysis of the mechanisms sustaining Pv transmission across this continent of low frequency of susceptible hosts found that reports of Pv prevalence were consistent with transmission being potentially limited to Duffy positive populations. Finally, reports of apparent Duffy-independent transmission are discussed. While Pv is evidently not a major malaria parasite across most of sub-Saharan Africa, the evidence presented here highlights its widespread low-level endemicity. An increased awareness of Pv as a potential malaria parasite, coupled with policy shifts towards species-specific diagnostics and reporting, will allow a robust assessment of the public health significance of Pv, as well as the other neglected non-Pf parasites, which are currently invisible to most public health authorities in Africa, but which can cause severe clinical illness and require specific control interventions.

Plasmodium falciparum encoded histidine rich protein (HRP2) based malaria rapid diagnostic tests (RDTs) are used in India. Deletion of pfhrp2 and pfhrp3 genes contributes to false negative test results, and large numbers of such deletions have been reported from South America, highlighting the importance of surveillance to detect such deletions.

Recent studies in Southeast Asia have demonstrated substantial zoonotic transmission of Plasmodium knowlesi to humans. Microscopically, P. knowlesi exhibits several stage-dependent morphological similarities to P. malariae and P. falciparum. These similarities often lead to misdiagnosis of P. knowlesi as either P. malariae or P. falciparum and PCR-based molecular diagnostic tests are required to accurately detect P. knowlesi in humans. The most commonly used PCR test has been found to give false positive results, especially with a proportion of P. vivax isolates. To address the need for more sensitive and specific diagnostic tests for the accurate diagnosis of P. knowlesi, we report development of a new single-step PCR assay that uses novel genomic targets to accurately detect this infection.

Primigravid (PG) women are at risk for pregnancy-associated malaria (PAM). Multigravid (MG) women acquire protection against PAM; however, HIV infection impairs this protective response. Protection against PAM is associated with the production of IgG specific for variant surface antigens (VSA-PAM) expressed by chondroitin sulfate A (CSA)-adhering parasitized erythrocytes (PEs). We hypothesized that VSA-PAM-specific IgG confers protection by promoting opsonic phagocytosis of PAM isolates and that HIV infection impairs this response.

Malaria transmission in African highland areas can be prone to epidemics, with minor fluctuations in temperature or altitude resulting in highly heterogeneous transmission. In the Kenyan Highlands, where malaria prevalence has been increasing, characterising malaria incidence and identifying risk factors for infection is complicated by asymptomatic infection.

Background: Individuals living in malaria-endemic regions develop immunity against severe malaria, but it is unclear whether immunity against pre-erythrocytic stages that blocks initiation of blood-stage infection after parasite inoculation develops following continuous natural exposure. Methods: We cleared schoolchildren living in an area (health district of Saponé, Burkina Faso) with highly endemic seasonal malaria of possible sub-patent infections and examined them weekly for incident infections by nested PCR. Plasma samples collected at enrolment were used to quantify antibodies to the pre-eryhrocytic-stage antigens circumsporozoite protein (CSP) and Liver stage antigen 1 (LSA-1). In vitro sporozoite gliding inhibition and hepatocyte invasion inhibition by naturally acquired antibodies were assessed using Plasmodium falciparum NF54 sporozoites. Associations between antibody responses, functional pre-erythrocytic immunity phenotypes and time to infection detected by 18S quantitative PCR were studied. Results: A total of 51 children were monitored. Anti-CSP antibody titres showed a positive association with sporozoite gliding motility inhibition (P<0.0001, Spearman's ρ=0.76). In vitro hepatocyte invasion was inhibited by naturally acquired antibodies (median inhibition, 19.4% [IQR 15.2-40.9%]), and there were positive correlations between invasion inhibition and gliding inhibition (P=0.005, Spearman's ρ=0.67) and between invasion inhibition and CSP-specific antibodies (P=0.002, Spearman's ρ=0.76). Survival analysis indicated longer time to infection in individuals displaying higher-than-median sporozoite gliding inhibition activity (P=0.01), although this association became non-significant after adjustment for blood-stage immunity (P = 0.06). Conclusions: In summary, functional antibodies against the pre-erythrocytic stages of malaria infection are acquired in children who are repeatedly exposed to Plasmodium parasites. This immune response does not prevent them from becoming infected during a malaria transmission season, but might delay the appearance of blood stage parasitaemia. Our approach could not fully separate the effects of pre-erythrocytic-specific and blood-stage-specific antibody-mediated immune responses in vivo; epidemiological studies powered and designed to address this important question should become a research priority.

Infection with Plasmodium can elicit antibodies that inhibit parasite survival in the mosquito, when they are ingested in an infectious blood meal. Here, we determine the transmission-reducing activity (TRA) of naturally acquired antibodies from 648 malaria-exposed individuals using lab-based mosquito-feeding assays. Transmission inhibition is significantly associated with antibody responses to Pfs48/45, Pfs230, and to 43 novel gametocyte proteins assessed by protein microarray. In field-based mosquito-feeding assays the likelihood and rate of mosquito infection are significantly lower for individuals reactive to Pfs48/45, Pfs230 or to combinations of the novel TRA-associated proteins. We also show that naturally acquired purified antibodies against key transmission-blocking epitopes of Pfs48/45 and Pfs230 are mechanistically involved in TRA, whereas sera depleted of these antibodies retain high-level, complement-independent TRA. Our analysis demonstrates that host antibody responses to gametocyte proteins are associated with reduced malaria transmission efficiency from humans to mosquitoes.

Intermittent preventive treatment (IPT) is a well established malaria control intervention. Evidence that delivering IPT to schoolchildren could provide community-level benefits is limited. We did a cluster-randomised controlled trial to assess the effect of IPT of primary schoolchildren with dihydroartemisinin-piperaquine (DP) on indicators of malaria transmission in the community, in Jinja, Uganda.

Single-dose primaquine (PQ) clears mature gametocytes and reduces the transmission of Plasmodium falciparum after artemisinin combination therapy. Genetic variation in CYP2D6, the gene producing the drug-metabolizing enzyme cytochrome P450 2D6 (CYP2D6), influences plasma concentrations of PQ and its metabolites and is associated with PQ treatment failure in Plasmodium vivax malaria. Using blood and saliva samples of varying quantity and quality from 8 clinical trials across Africa (n = 1,076), we were able to genotype CYP2D6 for 774 samples (72%). We determined whether genetic variation in CYP2D6 has implications for PQ efficacy in individuals with gametocytes at the time of PQ administration (n = 554) and for safety in glucose-6-phosphate dehydrogenase (G6PD)-deficient individuals treated with PQ (n = 110). Individuals with a genetically inferred CYP2D6 poor/intermediate metabolizer status had a higher gametocyte prevalence on day 7 or 10 after PQ than those with an extensive/ultrarapid CYP2D6 metabolizer status (odds ratio [OR] = 1.79 [95% confidence interval {CI}, 1.10, 2.90]; P = 0.018). The mean minimum hemoglobin concentrations during follow-up for G6PD-deficient individuals were 11.8 g/dl for CYP2D6 extensive/ultrarapid metabolizers and 12.1 g/dl for CYP2D6 poor/intermediate metabolizers (P = 0. 803). CYP2D6 genetically inferred metabolizer status was also not associated with anemia following PQ treatment (P = 0.331). We conclude that CYP2D6 poor/intermediate metabolizer status may be associated with prolonged gametocyte carriage after treatment with single-low-dose PQ but not with treatment safety.

Plasmodium falciparum and P. vivax co-exist at different endemicity levels across Ethiopia. For over two decades Artemether-Lumefantrine (AL) is the first line treatment for uncomplicated P. falciparum, while chloroquine (CQ) is still used to treat P. vivax. It is currently unclear whether a shift from CQ to AL for P. falciparum treatment has implications for AL efficacy and results in a reversal of mutations in genes associated to CQ resistance, given the high co-endemicity of the two species and the continued availability of CQ for the treatment of P. vivax. This study thus assessed the prevalence of Pfcrt-K76T and Pfmdr1-N86Y point mutations in P. falciparum. 18S RNA gene based nested PCR confirmed P. falciparum samples (N = 183) collected through community and health facility targeted cross-sectional surveys from settings with varying P. vivax and P. falciparum endemicity were used. The proportion of Plasmodium infections that were P. vivax was 62.2% in Adama, 41.4% in Babile, 30.0% in Benishangul-Gumuz to 6.9% in Gambella. The Pfcrt-76T mutant haplotype was observed more from samples with higher endemicity of P. vivax as being 98.4% (61/62), 100% (31/31), 65.2% (15/23) and 41.5% (22/53) in samples from Adama, Babile, Benishangul-Gumuz and Gambella, respectively. However, a relatively higher proportion of Pfmdr1-N86 allele (77.3-100%) were maintained in all sites. The observed high level of the mutant Pfcrt-76T allele in P. vivax co-endemic sites might require that utilization of CQ needs to be re-evaluated in settings co-endemic for the two species. A country-wide assessment is recommended to clarify the implication of the observed level of variation in drug resistance markers on the efficacy of AL-based treatment against uncomplicated P. falciparum malaria.

In 2004, in response to high levels of treatment failure associated with sulfadoxine-pyrimethamine (SP) resistance, Benin changed its first-line malaria treatment from SP to artemisinin-based combination therapy for treatment of uncomplicated Plasmodium falciparum malaria. Resistance to SP is conferred by accumulation of single nucleotide polymorphisms (SNPs) in P. falciparum genes involved in folate metabolism, dihydrofolate reductase (Pfdhfr) and dihydropteroate synthase (Pfdhps), targeted by pyrimethamine and sulfadoxine, respectively. Because SP is still used for intermittent preventive treatment in pregnant women (IPTp) and seasonal malaria chemoprevention (SMCP) in Benin, the prevalence of Pfdhfr and Pfdhps SNPs in P. falciparum isolates collected in 2017 were investigated.

Multiple red blood cell (RBC) variants appear to offer protection against the most severe forms of Plasmodium falciparum malaria. Associations between these variants and uncomplicated malaria are less clear.

The spread of drug resistance to antimalarial treatments poses a serious public health risk globally. To combat this risk, molecular surveillance of drug resistance is imperative. We report the prevalence of mutations in the Plasmodium falciparum kelch 13 propeller domain associated with partial artemisinin resistance, which we determined by using Sanger sequencing samples from patients enrolled in therapeutic efficacy studies from 9 sub-Saharan countries during 2014-2018. Of the 2,865 samples successfully sequenced before treatment (day of enrollment) and on the day of treatment failure, 29 (1.0%) samples contained 11 unique nonsynonymous mutations and 83 (2.9%) samples contained 27 unique synonymous mutations. Two samples from Kenya contained the S522C mutation, which has been associated with delayed parasite clearance; however, no samples contained validated or candidate artemisinin-resistance mutations.

Histidine-rich protein 2 (HRP2)-based rapid diagnostic tests detect Plasmodium falciparum malaria and are used throughout sub-Saharan Africa. However, deletions in the pfhrp2 and related pfhrp3 (pfhrp2/3) genes threaten use of these tests. Therapeutic efficacy studies (TESs) enroll persons with symptomatic P. falciparum infection. We screened TES samples collected during 2016-2018 in Ethiopia, Kenya, Rwanda, and Madagascar for HRP2/3, pan-Plasmodium lactate dehydrogenase, and pan-Plasmodium aldolase antigen levels and selected samples with low levels of HRP2/3 for pfhrp2/3 genotyping. We observed deletion of pfhrp3 in samples from all countries except Kenya. Single-gene deletions in pfhrp2 were observed in 1.4% (95% CI 0.2%-4.8%) of Ethiopia samples and in 0.6% (95% CI 0.2%-1.6%) of Madagascar samples, and dual pfhrp2/3 deletions were noted in 2.0% (95% CI 0.4%-5.9%) of Ethiopia samples. Although this study was not powered for precise prevalence estimates, evaluating TES samples revealed a low prevalence of pfhrp2/3 deletions in most sites.

Since 2005, artemisinin-based combination therapy (ACT) has been recommended to treat uncomplicated falciparum malaria in Madagascar. Artesunate-amodiaquine (ASAQ) and artemether-lumefantrine (AL) are the first- and second-line treatments, respectively. A therapeutic efficacy study was conducted to assess ACT efficacy and molecular markers of anti-malarial resistance.