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Asymptomatic malaria parasite carriers do not seek anti-malarial treatment and may constitute a silent infectious reservoir. In order to assess the level of asymptomatic and symptomatic carriage amongst adolescents in a highly endemic area, and to identify the risk factors associated with such carriage, we conducted a cross-sectional survey of 1032 adolescents (ages 10–19 years) from eight schools located in Ibadan, southwestern Nigeria in 2016. Blood films and blood spot filter paper samples were prepared for microscopy and DNA analysis. The prevalence of asymptomatic malaria was determined using microscopy, rapid diagnostic tests and PCR for 658 randomly selected samples. Of these, we found that 80% of asymptomatic schoolchildren were positive for malaria parasites by PCR, compared with 47% and 9%, determined by rapid diagnostic tests and microscopy, respectively. Malaria parasite species typing was performed using PCR targeting the mitochondrial CoxIII gene, and revealed high rates of carriage of Plasmodium malariae (53%) and Plasmodium ovale (24%). Most asymptomatic infections were co-infections of two or more species (62%), with Plasmodium falciparum + P. malariae the most common (35%), followed by P. falciparum + P. malariae + P. ovale (21%) and P. falciparum + P. ovale (6%). Single infections of P. falciparum, P. malariae and P. ovale accounted for 24%, 10% and 4% of all asymptomatic infections, respectively. To compare the species composition of asymptomatic and symptomatic infections, further sample collection was carried out in 2017 at one of the previously sampled schools, and at a nearby hospital. Whilst the species composition of the asymptomatic infections was similar to that observed in 2016, the symptomatic infections were markedly different, with single infections of P. falciparum observed in 91% of patients, P. falciparum + P. malariae in 5% and P. falciparum + P. ovale in 4%.

Malaria parasites complete their intra-erythrocytic developmental cycle (IDC) in multiples of 24 h suggesting a circadian basis, but the mechanism controlling this periodicity is unknown. Combining in vivo and in vitro approaches utilizing rodent and human malaria parasites, we reveal that: (i) 57% of Plasmodium chabaudi genes exhibit daily rhythms in transcription; (ii) 58% of these genes lose transcriptional rhythmicity when the IDC is out-of-synchrony with host rhythms; (iii) 6% of Plasmodium falciparum genes show 24 h rhythms in expression under free-running conditions; (iv) Serpentine receptor 10 (SR10) has a 24 h transcriptional rhythm and disrupting it in rodent malaria parasites shortens the IDC by 2-3 h; (v) Multiple processes including DNA replication, and the ubiquitin and proteasome pathways, are affected by loss of coordination with host rhythms and by disruption of SR10. Our results reveal malaria parasites are at least partly responsible for scheduling the IDC and coordinating their development with host daily rhythms., The mechanism underlying periodicity of Plasmodium’s intra-erythrocytic developmental cycle (IDC) is unclear. Here, Subudhi et al. show that serpentine receptor 10 (SR10) plays a role in regulating the schedule of the IDC in line with the timing of host daily rhythms.

Asymptomatic malaria parasite carriers do not seek anti-malarial treatment and may constitute a silent infectious reservoir. In order to assess the level of asymptomatic and symptomatic carriage amongst adolescents in a highly endemic area, and to identify the risk factors associated with such carriage, we conducted a cross sectional survey of 1032 adolescents (ages 10-19) from eight schools located in Ibadan, Southwest Nigeria in 2016. Blood films and blood spot filter paper samples were prepared for microscopy and DNA analysis. The prevalence of asymptomatic malaria was determined using microscopy, rapid diagnostic tests and PCR for 658 randomly selected samples. Of these, we found that 80% of asymptomatic schoolchildren were positive for malaria parasites by PCR, compared to 47% and 9% determined by RDT and microscopy, respectively. Malaria parasite species typing was performed using PCR targeting the mitochondrial CoxIII gene, and revealed high rates of carriage of Plasmodium malariae (53%) and Plasmodium ovale (24%). Most asymptomatic infections were co-infections of two or more species (62%), with P. falciparum + P. malariae the most common (35%), followed by P. falciparum + P. malariae + P. ovale (21%) and P. falciparum + P. ovale (6%). Single infections of P. falciparum, P. malariae and P. ovale accounted for 24%, 10% and 4% of all asymptomatic infections respectively. To compare the species composition of asymptomatic and symptomatic infections, further sample collection was carried out in 2017 at one of the previously sampled schools, and at a nearby hospital. Whilst the species composition of the asymptomatic infections was similar to that observed in 2016, the symptomatic infections were markedly different, with single infections of P. falciparum observed in 91% of patients, P. falciparum + P. malariae in 5% and P. falciparum + P. ovale in 4%.

Both vaccine and therapeutic approaches to malaria are based on conventional paradigms; whole organism or single antigen epitope-based vaccines administered with or without an adjuvant, and chemotherapeutics (anti-malaria drugs) that are toxic to the parasite. Two major problems that limit the effectiveness of these approaches are i) high levels of antigenic variation within parasite populations rendering vaccination efficacy against all variants difficult, and ii) the capacity of the parasite to quickly evolve resistance to drugs. We describe a new approach to both protection from and treatment of malaria parasites that involves the direct stimulation of the host innate immune response through the administration of a Toll-Like Receptor-2 (TLR2) agonist. The activity of PEG-Pam2Cys against the hepatocytic stages, erythrocytic stages and gametocytes of the rodent malaria parasite Plasmodium yoelii was investigated in laboratory mice. We show that administration of PEG-Pam2Cys, a soluble form of the TLR2 agonist S-[2,3-bis(palmitoyloxy)propyl] cysteine (Pam2Cys), significantly and dramatically reduces the numbers of malaria parasites that grow in the livers of mice following subsequent challenge with sporozoites. We also show that treatment can also clear parasites from the liver when administered subsequent to the establishment of infection. Finally, PEG-Pam2Cys can reduce the numbers of mosquitoes that are infected, and the intensity of their infection, following blood feeding on gametocytaemic mice. These results suggest that this compound could represent a novel liver stage anti-malarial that can be used both for the clearance of parasites following exposure and for the prevention of the establishment of infection., Graphical abstract Image 1, Highlights • TLR-2 agonist Pam2Cys reduces malaria parasite burden in the liver when administered prior to sporozoite challenge. • It also reduces malaria parasite burden in the liver when administered 24 h after sporozoite challenge. • It reduces the transmissibility of a malaria infection to mosquitoes.

Malaria parasites complete their intra-erythrocytic developmental cycle (IDC) in multiples of 24 hours (depending on the species), suggesting a circadian basis to the asexual cell cycle, but the mechanism controlling this periodicity is unknown. Combining in vivo and in vitro approaches using rodent and human malaria parasites, we reveal that: (i) 57% of Plasmodium chabaudi genes exhibit 24 h “circadian” periodicity in transcription; (ii) 58% of these genes lose transcriptional rhythmicity when the IDC is out-of-synchrony with host rhythms; (iii) 9% of Plasmodium falciparum genes show circadian transcription under free-running conditions; (iv) Serpentine receptor 10 (SR10) has a circadian transcription profile and disrupting it in rodent malaria parasites shortens the IDC by 2-3 hours; (v) Multiple processes including DNA replication and the ubiquitin and proteasome pathways are affected by loss of coordination with host rhythms and by disruption of SR10. Our results show that malaria parasites are at least partly responsible for scheduling their IDCs explaining the fitness benefits of coordination with host rhythms.

Following the bite of an infective mosquito, malaria parasites first invade the liver where they develop and replicate for a number of days before being released into the bloodstream where they invade red blood cells and cause disease. The biology of the liver stages of malaria parasites is relatively poorly understood due to the inaccessibility of the parasites to sampling during this phase of their life cycle. Here we report the detection in blood and faecal samples of malaria parasite DNA throughout their development in the livers of mice and before the parasites begin their growth in the blood circulation. It is shown that parasite DNA derived from pre-erythrocytic stage parasites reaches the faeces via the bile. We then show that different primate malaria species can be detected by PCR in blood and faecal samples from naturally infected captive macaque monkeys. These results demonstrate that pre-erythrocytic parasites can be detected and quantified in experimentally infected animals. Furthermore, these results have important implications for both molecular epidemiology and phylogenetics of malaria parasites. In the former case, individuals who are malaria parasite negative by microscopy, but PCR positive for parasite DNA in their blood, are considered to be "sub-microscopic" blood stage parasite carriers. We now propose that PCR positivity is not necessarily an indicator of the presence of blood stage parasites, as the DNA could derive from pre-erythrocytic parasites. Similarly, in the case of molecular phylogenetics based on DNA sequences alone, we argue that DNA amplified from blood or faeces does not necessarily come from a parasite species that infects the red blood cells of that particular host.

In endemic areas with high transmission intensities, malaria infections are very often composed of multiple genetically distinct strains of malaria parasites. It has been hypothesised that this leads to intra-host competition, in which parasite strains compete for resources such as space and nutrients. This competition may have repercussions for the host, the parasite, and the vector in terms of disease severity, vector fitness, and parasite transmission potential and fitness. It has also been argued that within-host competition could lead to selection for more virulent parasites. Here we use the rodent malaria parasite Plasmodium yoelii to assess the consequences of mixed strain infections on disease severity and parasite fitness. Three isogenic strains with dramatically different growth rates (and hence virulence) were maintained in mice in single infections or in mixed strain infections with a genetically distinct strain. We compared the virulence (defined as harm to the mammalian host) of mixed strain infections with that of single infections, and assessed whether competition impacted on parasite fitness, assessed by transmission potential. We found that mixed infections were associated with a higher degree of disease severity and a prolonged infection time. In the mixed infections, the strain with the slower growth rate was often responsible for the competitive exclusion of the faster growing strain, presumably through host immune-mediated mechanisms. Importantly, and in contrast to previous work conducted with Plasmodium chabaudi, we found no correlation between parasite virulence and transmission potential to mosquitoes, suggesting that within-host competition would not drive the evolution of parasite virulence in P. yoelii., Author Summary Malaria infections are very often composed of multiple strains of malaria parasites. It is thought that these strains may compete for resources such as space and nutrients within the host. Here we show that such “within-host competition” has repercussions for the virulence of the malaria infection, so that infections composed of multiple strains are more virulent in terms of disease severity, than single strain infections containing the constituent parasites. Following from this, it has been proposed that as such competition would favour those parasites with faster growth rates, then these parasites would be selected in nature when within-host competition is common. We show, however, that this is not necessarily the case, as parasites with faster growth rates in the mammalian host were no more successful at transmitting to mosquitoes than parasites with slower growth rates. These results show that a reassessment of our current understanding of the role of within-host competition in the selection of virulence in malaria parasites is required.

Identifying the genetic determinants of phenotypes that impact disease severity is of fundamental importance for the design of new interventions against malaria. Here we present a rapid genome-wide approach capable of identifying multiple genetic drivers of medically relevant phenotypes within malaria parasites via a single experiment at single gene or allele resolution. In a proof of principle study, we found that a previously undescribed single nucleotide polymorphism in the binding domain of the erythrocyte binding like protein (EBL) conferred a dramatic change in red blood cell invasion in mutant rodent malaria parasites Plasmodium yoelii. In the same experiment, we implicated merozoite surface protein 1 (MSP1) and other polymorphic proteins, as the major targets of strain-specific immunity. Using allelic replacement, we provide functional validation of the substitution in the EBL gene controlling the growth rate in the blood stages of the parasites., Author summary Developing a greater understanding of malaria genetics is a key step in combating the threat posed by the disease. Here we use a novel approach to study two important properties of the parasite; the rate at which parasites grow within a single host, and the means by which parasites are affected by the host immune system. Two malaria strains with different biological properties were crossed in mosquitoes to produce a hybrid population, which was then grown in naïve and vaccinated mice. Parasites with genes conveying increased growth or immune evasion are favoured under natural selection, leaving a signature on the genetic composition of the cross population. We describe a novel mathematical approach to interpret this signature, identifying selected genes within the parasite population. We discover new genetic variants conveying increased within-host growth and resistance to host immunity in a mouse malaria strain. Experimental validation highlights the ability of this rapid experimental process for generating insights into malaria biology.