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Human malaria continues to be a public health problem and an important cause of morbidity and mortality in the world. Malaria control is achieved through both individual protection against mosquito bites and drug treatment, which is hampered by the spread of Plasmodium falciparum resistance to most antimalarials, including artemisinin derivatives. One of the key pharmacological strategies for controlling malaria is to block transmission of the parasites to their mosquito vectors. Following this rational, MEFAS, a synthetic hybrid salt derived from artesunate (AS) and mefloquine has been previously reported for its activity against asexual P. falciparum parasites in vitro, in addition to a pronounced reduction in the viability of mature gametocytes. Herein, MEFAS was tested against asexual forms of Plasmodium vivax and for its ability to block malaria transmission in Anopheles darlingi mosquitoes in a membrane feeding assay using P. vivax field isolates. MEFAS demonstrated high potency, with a IC50 of 6.5 nM against asexual forms of P. vivax. At 50 μM, MEFAS completely blocked oocyst formation in mosquitoes, regardless of the oocyst number in the control group. At lower doses, MEFAS reduced oocyst prevalence by greater than 20%. At equivalent doses, AS irregularly reduced oocyst formation and caused only slight inhibition of mosquito infections. These results highlight the potential of MEFAS as a novel transmission-blocking molecule, as well as its high blood schizonticidal activity against P. vivax and P. falciparum field isolates, representing a starting point for further development of a new drug with dual antimalarial activity., Graphical abstract Image 1

Haemosporidian parasites are responsible for anemia, acute tissue degeneration, and depopulation in wild birds. This study aimed to investigate the prevalence of haemosporidians and also morphologic and molecular evaluation of tissue stages of Haemoproteus sp. in common buzzards (Buteo buteo). Eleven free-living common buzzards were referred to the Avian Clinic of Veterinary School of Lorestan University with lethargy, weight loss, and ataxia. Gametocytes of Leucocytozoon buteonis were found in blood smears of six (54.5 %) birds, while one had simultaneous infection with blood stages of Haemoproteus and Leucocytozoon. During histopathological examinations, exo-erythrocytic stages of the genus Haemoproteus were seen in the lung and kidney of a dead bird. This study is the first report of exo-erythrocytic infection of Haemoproteus in common buzzards. Molecular assays confirmed the infection of Haemoproteus sp. (lineage BUTBUT15) in tissue samples. Phylogenetic analysis using cytochrome b gene suggested that BUTBUT15 was more closely related to the lineages isolated from the family Falconidae in contrast to the Accipitridae.

Background Plasmodium interspersed repeat (pir) is the largest multigene family in the genomes of most Plasmodium species. A variety of functions for the PIR proteins which they encode have been proposed, including antigenic variation, immune evasion, sequestration and rosetting. However, direct evidence for these is lacking. The repetitive nature of the family has made it difficult to determine function experimentally. However, there has been some success in using gene expression studies to suggest roles for some members in virulence and chronic infection. Methods Here pir gene expression was examined across the life cycle of Plasmodium berghei using publicly available RNAseq data-sets, and at high resolution in the intraerythrocytic development cycle using new data from Plasmodium chabaudi. Results Expression of pir genes is greatest in stages of the parasite which invade and reside in red blood cells. The marked exception is that liver merozoites and male gametocytes produce a very large number of pir gene transcripts, notably compared to female gametocytes, which produce relatively few. Within the asexual blood stages different subfamilies peak at different times, suggesting further functional distinctions. Representing a subfamily of its own, the highly conserved ancestral pir gene warrants further investigation due to its potential tractability for functional investigation. It is highly transcribed in multiple life cycle stages and across most studied Plasmodium species and thus is likely to play an important role in parasite biology. Conclusions The identification of distinct expression patterns for different pir genes and subfamilies is likely to provide a basis for the design of future experiments to uncover their function.

Antimalarial drugs capable of targeting multiple parasite stages, particularly the transmissible stages, can be valuable tools for advancing the malaria elimination agenda. Current antifolate drugs such as pyrimethamine can inhibit replicative parasite stages in both humans and mosquitoes, but antifolate resistance remains a challenge. The lack of reliable gametocyte-producing, antifolate-resistant Plasmodium falciparum laboratory strain hinders the study of new antifolate compounds that can overcome antifolate resistance including development stages in the mosquito. We used clustered regularly interspaced short palindromic repeats-Cas9 genome editing to develop a transgenic gametocyte-producing strain of P. falciparum with quadruple mutations (N51I, C59R, S108N, I164L) in the dihydrofolate reductase (dhfr) gene, using NF54 as a parental strain. The transgenic parasites exhibited pyrimethamine resistance while maintaining their gametocyte-producing activity. We then demonstrated that pyrimethamine could no longer inhibit male gametocyte exflagellation in the transgenic parasite. In contrast, P218, the novel antifolate, designed to overcome antifolate resistance, potently inhibited exflagellation. The exflagellation IC50 of P218 was five times lower than the asexual stage half maximal inhibitory concentration (IC50), suggesting a strong barrier for transmission of P218-resistant parasites. The transgenic gametocyte-producing, pyrimethamine-resistant parasite is a robust system for evaluating novel antifolate compounds against non-asexual stage development.

Assays used to evaluate the transmission-blocking activity of antimalarial drugs are largely focused on their potential to inhibit or reduce the infectivity of gametocytes, the blood stages of the parasite that are responsible for the onward transmission to the mosquito vector. For this purpose, the drug is administered concomitantly with gametocyte-infected blood, and the results are evaluated as the percentage of reduction in the number of oocysts in the mosquito midgut. We report the results of a series of experiments that explore the transmission-blocking potential of two key antimalarial drugs, artesunate and sulfadoxine-pyrimethamine, when administered to mosquitoes already infected from a previous blood meal. For this purpose, uninfected mosquitoes and mosquitoes carrying a 6 day old Plasmodium relictum infection (early oocyst stages) were allowed to feed either on a drug-treated or an untreated host in a fully factorial experiment. This protocol allowed us to bypass the gametocyte stages and establish whether the drugs have a sporontocidal effect, i.e. whether they are able to arrest the ongoing development of oocysts and sporozoites, as would be the case when a mosquito takes a post-infection treated blood meal. In a separate experiment, we also explored whether a drug-treated blood meal impacted key life history traits of the mosquito relevant for transmission, and if this depended on their infection status. Our results showed that feeding on an artesunate- or sulfadoxine-pyrimethamine-treated hosts has no epidemiologically relevant effects on the fitness of infected or uninfected mosquitoes. In contrast, when infected mosquitoes fed on an sulfadoxine-pyrimethamine-treated host, we observed both a significant increase in the number of oocysts in the midgut, and a drastic decrease in both sporozoite prevalence (−30%) and burden (−80%) compared with the untreated controls. We discuss the potential mechanisms underlying these seemingly contradictory results and contend that, provided the results are translatable to human malaria, the potential epidemiological and evolutionary consequences of the current preventive use of sulfadoxine-pyrimethamine in malaria-endemic countries could be substantial.

Background Malaria control in sub-Saharan Africa relies upon prompt case management with artemisinin-based combination therapy (ACT). Ring-stage parasite mRNA, measured by sbp1 quantitative reverse-transcriptase PCR (qRT-PCR), was previously reported to persist after ACT treatment and hypothesized to reflect temporary arrest of the growth of ring-stage parasites (dormancy) following exposure to artemisinins. Here, the persistence of ring-stage parasitaemia following ACT and non-ACT treatment was examined. Methods Samples were used from naturally infected Malian gametocyte carriers who received dihydroartemisinin–piperaquine (DP) or sulfadoxine–pyrimethamine (SP–AQ) with or without gametocytocidal drugs. Gametocytes and ring-stage parasites were quantified by qRT-PCR during 42 days of follow-up. Results At baseline, 89% (64/73) of participants had measurable ring-stage parasite mRNA. Following treatment, the proportion of ring-stage parasite-positive individuals and estimated densities declined for all four treatment groups. Ring-stage parasite prevalence and density was generally lower in arms that received DP compared to SP–AQ. This finding was most apparent days 1, 2, and 42 of follow-up (p day 14 = 0.011 and pday 28 = 0.068). No association of ring-stage persistence with gametocyte carriage was observed. Conclusions The current findings of lower ring-stage persistence after ACT without an effect of gametocytocidal partner drugs affirms the use of sbp1 as ring-stage marker. Lower persistence of ring-stage mRNA after ACT treatment suggests the marker may not reflect dormant parasites whilst it was predictive of re-appearance of parasitaemia.

Parasites of the haemosporidian genus Polychromophilus have exclusively been described in bats. These parasites belong to the diverse group of malaria parasites, and Polychromophilus presents the only haemosporidian taxon that infects mammalian hosts in tropical as well as in temperate climate zones. This study provides the first information of Polychromophilus parasites in the lesser Asiatic yellow bat (Scotophilus kuhlii) in Thailand, a common vespertilionid bat species distributed in South and Southeast Asia. The gametocyte blood stages of the parasites could not be assigned to a described morphospecies and molecular analysis revealed that these parasites might represent a distinct Polychromophilus species. In contrast to Plasmodium species, Polychromophilus parasites do not multiply in red blood cells and, thus, do not cause the clinical symptoms of malaria. Parasitological and molecular investigation of haemosporidian parasites of wildlife, such as the neglected genus Polychromophilus, will contribute to a better understanding of the evolution of malaria parasites.

The cold shock domain (CSD) forms the hallmark of the cold shock protein family that provides the characteristic feature of binding with nucleic acids. While much of the information is available on bacterial, plants and human cold shock proteins, their existence and functions in the malaria parasite remains undefined. In the present review, the available information on functions of well-characterized cold shock protein members in different organisms has been collected and an attempt was made to identify the presence and role of cold shock proteins in malaria parasite. A singlePlasmodium falciparumcold shock protein (PfCoSP) was found inP. falciparumwhich is reported to be essential for parasite survival. Essentiality ofPfCoSPunderscores its importance in malaria parasite life cycle. In silico tools were used to predict the features ofPfCoSPand to identify its homologues in bacteria, plants, humans, and otherPlasmodiumspecies. Modelled structures ofPfCoSPand its homologues inPlasmodiumspecies were compared with human cold shock protein ‘YBOX-1’ (Y-box binding protein 1) that provide important insights into their functioning.PfCoSPmodel was subjected to docking with B-form DNA and RNA to reveal a number of residues crucial for their interaction. Transcriptome analysis and motifs identified inPfCoSPimplicate its role in controlling gene expression at gametocyte, ookinete and asexual blood stages of malaria parasite. Overall, this review emphasizes the functional diversity of the cold shock protein family by discussing their known roles in gene expression regulation, cold acclimation, developmental processes like flowering transition, and flower and seed development, and probable function in gametocytogenesis in case of malaria parasite. This enables readers to view the cold shock protein family comprehensively.

Background To understand the dynamics of malaria transmission, membrane feeding assays with glass feeders are used to assess the transmission potential of malaria infected individuals to mosquitoes. However, in some circumstances, use of these assays is hindered by both the blood volume requirement and the availability of fragile, specially crafted glass feeders. 3D printed plastic feeders that require very small volumes of blood would thus expand the utility of membrane feeding assays. Methods Using two 3D printing production methods, MultiJet (MJ) and Digital Light Processing (DLP), we developed a plastic version of the most commonly used standard glass feeder (the mini-feeder) with an improved design, and also a smaller feeder requiring only 60 µl of blood (the nano-feeder). Performance of the 3D printed feeders was compared to standard glass mini-feeders by assessing infectivity of gametocytes to mosquitoes in standard membrane feeding assays with laboratory reared Anopheles stephensi mosquitoes and cultured Plasmodium falciparum gametocytes. In addition, the optimum number of mosquitoes that can feed on the nano-feeder was determined by evaluating fully fed mosquitoes visually and by assessing blood- meal volume with a colorimetric haemoglobin assay. Results The 3D printing methods allowed quick and inexpensive production of durable feeders. Infectivity of gametocytes to mosquitoes was comparable for MJ and DLP 3D printed feeders and glass feeders, and the performance of the 3D printed feeders was not influenced by repeated washing with bleach. There was no loss in transmission efficiency when the feeder size was reduced from mini-feeder to nano-feeder, and blood-meal volume assessment indicated ~10 An. stephensi mosquitoes can take a full blood-meal (median volume 3.44 µl) on a nano-feeder. Conclusions Here we present 3D printed mini- and nano-feeders with comparable performance to the currently used glass mini-feeders. These feeders do not require specialized glass craftsmanship, making them easily accessible. Moreover, the smaller nano-feeders will enable evaluation of smaller blood volumes that can be collected from finger prick, thus expanding the utility of membrane feeding assays and facilitating a more thorough evaluation of the human infectious reservoir for malaria.

The discovery and development of multistage antimalarial drugs targeting intra-erythrocytic asexual and sexual Plasmodium falciparum parasites is of utmost importance to achieve the ambitious goal of malaria elimination. Here, we report the validation of naphthylisoquinoline (NIQ) alkaloids and their synthetic analogues as multistage active antimalarial drug candidates. A total of 30 compounds were tested, of which 17 exhibited IC50 values 10. The tested compounds showed activity in vitro against both early- and late-stage P. falciparum gametocytes while blocking male gamete formation (>70% inhibition of exflagellation at 2 μM). Additionally, five selected compounds were found to have good solubility (≥170 μM in PBS at pH 6.5), while metabolic stability towards human, mouse, and rat microsomes ranged from >90% to >7% after 30 min. Dioncophylline C (2a) emerged as a front runner from the study, displaying activity against both asexual parasites and gametocytes, a lack of cross-resistance to chloroquine, good solubility, and microsomal stability. Overall, this is the first report on the multistage activity of NIQs and their synthetic analogues including gametocytocidal and gametocidal effects induced by this class of compounds., Graphical abstract Image 1, Highlights • Naphthylisoquinolines (NIQs) validated as antimalarial hit candidates. • First report on transmission-blocking properties of NIQs and analogues. • 15 compounds active across 9 P. falciparum strains, with acceptable RI 10. • 5 compounds show good solubility and microsomal stability. • Dioncophylline C is the frontrunner antimalarial candidate with multistage activity.

Background PCR can be positive weeks after effective malaria treatment, potentially leading to over diagnose of recrudescence and re-infections. The DNA detected by PCR post-treatment might stem from residuals of destroyed asexual parasites, or from live gametocytes. The objective of this clinical observational study was to describe the presence of positive PCR for Plasmodium falciparum and Plasmodium vivax in follow-up samples post-treatment from returned travellers, and the proportion of positive PCR due to gametocytes. Methods Whole blood was collected during hospitalization and outpatient routine follow-up from 13 patients with imported malaria. DNA was extracted applying QIAamp DNA Blood Mini Kit, while mRNA was collected and extracted applying PAXgene Blood RNA Tubes and Kit. All DNA samples (N = 25) were analysed with a genus-specific cytb real-time SYBR PCR, and P. falciparum DNA samples (N = 22) were also analysed with a falciparum-specific varATS real-time TaqMan PCR. All the mRNA samples (N = 18) were analysed with both a genus-specific 18S rRNA RT-PCR and a gametocyte-specific Pfs25 (P. falciparum)/Pvs25 (P. vivax) RT-PCR. Results Latest samples were collected at day 1 (n = 2) and from day 11–54 (n = 11) after treatment. Genus DNA cytb PCR was positive up to 49 days after effective treatment, and 18S rRNA transcripts from active P. falciparum parasites were detectable for at least 11 days. Gametocyte-specific mRNA was detected at latest only two days after treatment. Among six patients with late positive PCR for P. falciparum, four had high parasitaemia at admittance (6–30%), while two had parasitaemia P. vivax was not found by any of the PCR methods. Conclusions DNA-based PCR can be positive up to at least seven weeks after curative malaria treatment, potentially leading to over-diagnose of recrudescence and re-infections. Based on the observations in this study, it is unclear if the DNA origins from residuals of destroyed parasites or live gametocytes, warranting further investigations.

Coccidian parasites possess complex life cycles involving asexual proliferation followed by sexual development leading to the production of oocysts. Coccidian oocysts are persistent stages which are secreted by the feces and transmitted from host to host guaranteeing life cycle progression and disease transmission. The robust bilayered oocyst wall is formed from the contents of two organelles, the wall-forming bodies type I and II (WFBI, WFBII), located exclusively in the macrogametocyte. Eimeria nieschulzi has been used as a model parasite to study and follow gametocyte and oocyst development. In this study, the gametocyte and oocyst wall formation of E. nieschulzi was analyzed by electron microscopy and immuno-histology. A monoclonal antibody raised against the macrogametocytes of E. nieschulzi identified a tyrosine-rich glycoprotein (EnGAM82) located in WFBII. Correlative light and electron microscopy was used to examine the vesicle-specific localization and spatial distribution of GAM82-proteins during macrogametocyte maturation by this monoclonal antibody. In early and mid-stages, the GAM82-protein is ubiquitously distributed in WFBII. Few hours later, the protein is arranged in subvesicular structures. It was possible to show that the substructure of WFBII and the spatial distribution of GAM82-proteins probably represent pre-synthesized cross-linked materials prior to the inner oocyst wall formation. Dityrosine-cross-linked gametocyte proteins can also be confirmed and visualized by fluorescence microscopy (UV light, autofluorescence of WFBII). Electronic supplementary material The online version of this article (10.1007/s00436-020-06765-6) contains supplementary material, which is available to authorized users.

Background Red blood cell (RBC) polymorphisms are suggested to influence the course of Plasmodium falciparum malaria. Whereas some variants have been found to be protective, others have been found to enhance parasite development. This study evaluated the effect of variant haemoglobin (Hb) and ABO blood groups on P. falciparum merozoite invasion, multiplication rates as well as gametocyte development. Methods Approximately 2.5 mL of venous blood was collected from each participant. Flow cytometry was used to determine the in vitro merozoite invasion rates of NF54 parasites into the blood of 66 non-parasitaemic individuals with variant Hb genotypes (HbSS, HbSC) and blood groups (A, B, O), which were then compared with invasion into HbAA blood. The ex vivo asexual parasite multiplication and gametocyte production rates of parasites from 79 uncomplicated malaria patients with varying Hb genotypes (HbAS, HbAC and HbAA) were also estimated using microscopy. Results Merozoite invasion rates were significantly reduced by about 50% in RBCs containing HbSS and HbSC relative to HbAA cells. The presence of blood group O and B reduced the invasion rates of HbSS by about 50% and 60%, respectively, relative to HbSC but the presence of blood group A removed the inhibitory effect of HbSS. The initial parasite densities in uncomplicated malaria patients with Hb genotypes HbAS and HbAC cells were similar but significantly lower than those with genotype HbAA. The ex vivo parasite multiplication rate, gametocytaemia and gametocyte conversion rates followed a similar trend but did not reach statistical significance (p > 0.05). Conclusions Parasite invasion rate into erythrocytes is dependent on both erythrocyte blood group antigen and haemoglobin genotype as blood group O and B provided protection via reduced merozoite invasion in RBCs containing HbSS relative to HbSC. Regardless of haemoglobin type, greater than 70% malaria patients had circulating ring stage parasites that differentiated into stage II gametocytes in 4 days.

Background Plasmodium falciparum zygotes develop in the mosquito midgut after an infectious blood meal containing mature male and female gametocytes. Studies of mosquito-produced P. falciparum zygotes to elucidate their biology and development have been hampered by high levels of contaminating mosquito proteins and macromolecules present in zygote preparations. Thus, no zygote-specific surface markers have been identified to date. Here, a methodology is developed to obtain large quantities of highly purified zygotes using in vitro culture, including purification methods that include magnetic column cell separation (MACS) followed by Percoll density gradient centrifugation. This straightforward and effective approach provides ample material for studies to enhance understanding of zygote biology and identify novel zygote surface marker candidates that can be tested as transmission blocking vaccine (TBV) candidates. Methods Plasmodium falciparum gametocyte cultures were established and maintained from asexual cultures. Gametocytes were matured for 14 days, then transferred into zygote media for 6 h at 27 ± 2 °C to promote gamete formation and fertilization. Zygotes were then purified using a combination of MACS column separation and Percoll density gradient centrifugation. Purity of the zygotes was determined through morphological studies: the parasite body and nuclear diameter were measured, and zygotes were further transformed into ookinetes. Immunofluorescence assays (IFA) were also performed using the ookinete surface marker, Pfs28. Results After stimulation, the culture consisted of transformed zygotes and a large number of uninfected red blood cells (RBCs), as well as infected RBCs with parasites at earlier developmental stages, including gametes, gametocytes, and asexual stages. The use of two MACS columns removed the vast majority of the RBCs and gametocytes. Subsequent use of two Percoll density gradients enabled isolation of a pure population of zygotes. These zygotes transformed into viable ookinetes that expressed Pfs28. Conclusion The combined approach of using two MACS columns and two Percoll density gradients yielded zygotes with very high purity (45-fold enrichment and a pure population of zygotes [approximately 100%]) that was devoid of contamination by other parasite stages and uninfected RBCs. These enriched zygotes, free from earlier parasites stages and mosquito-derived macromolecules, can be used to further elucidate the biology and developmental processes of Plasmodium.

Background Mosquito-feeding assays that assess transmission of Plasmodium from man-to-mosquito typically use laboratory mosquito colonies. The microbiome and genetic background of local mosquitoes may be different and influence Plasmodium transmission efficiency. In order to interpret transmission studies to the local epidemiology, it is therefore crucial to understand the relationship between infectivity in laboratory-adapted and local mosquitoes. Methods We assessed infectivity of Plasmodium vivax-infected patients from Adama, Ethiopia, using laboratory-adapted (colony) and wild-caught (wild) mosquitoes raised from larval collections in paired feeding experiments. Feeding assays used 4–6 day-old female Anopheles arabiensis mosquitoes after starvation for 12 h (colony) and 18 h (wild). Oocyst development was assessed microscopically 7 days post-feeding. Wild mosquitoes were identified morphologically and confirmed by genotyping. Asexual parasites and gametocytes were quantified in donor blood by microscopy. Results In 36 paired experiments (25 P. vivax infections and 11 co-infections with P. falciparum), feeding efficiency was higher in colony (median: 62.5%; interquartile range, IQR: 47.0–79.0%) compared to wild mosquitoes (median: 27.8%; IQR: 17.0–38.0%; Z = 5.02; P < 0.001). Plasmodium vivax from infectious individuals (51.6%, 16/31) infected a median of 55.0% (IQR: 6.7–85.7%; range: 5.5–96.7%; n = 14) of the colony and 52.7% (IQR: 20.0–80.0%; range: 3.2–95.0%; n = 14) of the wild mosquitoes. A strong association (ρ(16) = 0.819; P < 0.001) was observed between the proportion of infected wild and colony mosquitoes. A positive association was detected between microscopically detected gametocytes and the proportion of infected colony (ρ(31) = 0.452; P = 0.011) and wild (ρ(31) = 0.386; P = 0.032) mosquitoes. Conclusions Infectivity assessments with colony and wild mosquitoes yielded similar infection results. This finding supports the use of colony mosquitoes for assessments of the infectious reservoir for malaria in this setting whilst acknowledging the importance of mosquito factors influencing sporogonic development of Plasmodium parasites.

Background Asymptomatic carriage of Plasmodium falciparum is widespread in adults and children living in malaria-endemic countries. This study identified the prevalence of malaria parasites and the corresponding levels of naturally acquired anti-parasite antibody levels in afebrile adults living in two communities in the Greater Accra Region of Ghana. Methods Two cross-sectional studies conducted in January and February 2016 and repeated in July and August 2016 recruited subjects aged between 6 and 75 years from high parasite prevalence (Obom) and low parasite prevalence (Asutsuare) communities. Whole blood (5 ml) was collected from each volunteer, plasma was aliquoted and frozen until needed. An aliquot (10 µl) of the blood was used to prepare thick and thin blood smears, 100 µl was preserved in Trizol and the rest was separated into plasma and blood cells and each stored at − 20 °C until needed. Anti-MSP3 and Pfs230 antibody levels were measured using ELISA. Results Asexual parasite and gametocyte prevalence were higher in Obom than Asutsuare. Antibody (IgG, IgG1, IgG3, IgM) responses against the asexual parasite antigen MSP3 and gametocyte antigen Pfs230 were higher in Obom during the course of the study except for IgM responses against Pfs230, which was higher in Asutsuare than in Obom during the rainy season. Antibody responses in Asutsuare were more significantly associated with age than the responses measured in Obom. Conclusion The pattern of antibody responses measured in people living in the high and low malaria transmission setting was similar. All antibody responses measured against the asexual antigen MSP3 increased, however, IgG and IgG1 responses against gametocyte antigen Pfs230 decreased in moving from the dry to the peak season in both sites. Whilst asexual and gametocyte prevalence was similar between the seasons in the low transmission setting, in the high transmission setting asexual parasite prevalence increased but gametocyte prevalence decreased in the rainy season relative to the dry season.

Background The intraerythrocytic development cycle (IDC) of the rodent malaria Plasmodium chabaudi is coordinated with host circadian rhythms. When this coordination is disrupted, parasites suffer a 50% reduction in both asexual stages and sexual stage gametocytes over the acute phase of infection. Reduced gametocyte density may not simply follow from a loss of asexuals because investment into gametocytes (“conversion rate”) is a plastic trait; furthermore, the densities of both asexuals and gametocytes are highly dynamic during infection. Hence, the reasons for the reduction of gametocytes in infections that are out-of-synch with host circadian rhythms remain unclear. Here, two explanations are tested: first, whether out-of-synch parasites reduce their conversion rate to prioritize asexual replication via reproductive restraint; second, whether out-of-synch gametocytes experience elevated clearance by the host’s circadian immune responses. Methods First, conversion rate data were analysed from a previous experiment comparing infections of P. chabaudi that were in-synch or 12 h out-of-synch with host circadian rhythms. Second, three new experiments examined whether the inflammatory cytokine TNF varies in its gametocytocidal efficacy according to host time-of-day and gametocyte age. Results There was no evidence that parasites reduce conversion or that their gametocytes become more vulnerable to TNF when out-of-synch with host circadian rhythms. Conclusions The factors causing the reduction of gametocytes in out-of-synch infections remain mysterious. Candidates for future investigation include alternative rhythmic factors involved in innate immune responses and the rhythmicity in essential resources required for gametocyte development. Explaining why it matters for gametocytes to be synchronized to host circadian rhythms might suggest novel approaches to blocking transmission.

Background The efficacy and safety of artemether–lumefantrine (AL) and dihydroartemisinin–piperaquine (DP) against asexual parasites population has been documented. However, the effect of these anti-malarials on sexual parasites is still less clear. Gametocyte clearance following treatment is essential for malaria control and elimination efforts; therefore, the study sought to determine trends in gametocyte clearance after AL or DP treatment in children from a malaria-endemic site in Kenya. Methods Children aged between 0.5 and 12 years from Busia, western Kenya with uncomplicated Plasmodium falciparum malaria were assigned randomly to AL or DP treatment. A total of 334 children were enrolled, and dried blood spot samples were collected for up to 6 weeks after treatment during the peak malaria transmission season in 2016 and preserved. Plasmodium falciparum gametocytes were detected by qRT-PCR and gametocyte prevalence, density and mean duration of gametocyte carriage were determined. Results At baseline, all the 334 children had positive asexual parasites by microscopy, 12% (40/334) had detectable gametocyte by microscopy, and 83.7% (253/302) children had gametocytes by RT-qPCR. Gametocyte prevalence by RT-qPCR decreased from 85.1% (126/148) at day 0 to 7.04% (5/71) at day 42 in AL group and from 82.4% (127/154) at day 0 to 14.5% (11/74) at day 42 in DP group. The average duration of gametocyte carriage as estimated by qRT-PCR was slightly shorter in the AL group (4.5 days) than in the DP group (5.1 days) but not significantly different (p = 0.301). Conclusion The study identifies no significant difference between AL and DP in gametocyte clearance. Gametocytes persisted up to 42 days post treatment in minority of individuals in both treatment arms. A gametocytocidal drug, in combination with artemisinin-based combination therapy, will be useful in blocking malaria transmission more efficiently.

Background Haemosporidian parasites of the genus Haemoproteus (Haemoproteidae) are widespread and cause haemoproteosis in birds and therefore, their diversity, ecology and evolutionary biology have become subjects of intensive research. However, the vectors and transmission patterns of haemoproteids as well as the epidemiology of haemoproteosis remain insufficiently investigated. Several species of Culicoides (Ceratopogonidae) support complete sporogony of haemoproteids belonging to the subgenus Parahaemoproteus. However, experimental research with these fragile insects is difficult to design in the field, particularly because their abundance markedly depends on seasonality. This is an obstacle for continuous sampling of sufficient numbers of naturally infected or experimentally exposed midges from wildlife. We developed simple methodology for accessing sporogonic development of haemoproteids in laboratory-reared Culicoides nubeculosus. This study aimed to describe the mosaic of methods constituting this methodology, which was applied for investigation of the sporogonic development of Haemoproteus (Parahaemoproteus) pastoris, a widespread parasite of the common starling Sturnus vulgaris. Methods The methodology consists of the following main stages: (i) laboratory rearing of C. nubeculosus from the egg stage to adult insects; (ii) selection of naturally infected birds, the donors of mature gametocytes to expose biting midges; (iii) experimental exposure of insects and their laboratory maintenance; and (iv) dissection of exposed insects. Biting midges were exposed to H. pastoris (cytochrome b lineage hLAMPUR01) detected in one naturally infected common starling. Engorged insects were dissected at intervals in order to follow sporogony. Microscopic examination and PCR-based methods were used to identify the sporogonic stages and to confirm the presence of the parasite lineage in infected insects, respectively. Results Culicoides nubeculosus females were successfully reared and exposed to H. pastoris, which completed sporogonic development 7–9 days post-infection when sporozoites were observed in the salivary glands. Conclusions The new methodology is easy to use and non-harmful for birds, providing opportunities to access the sporogonic stages of Parahaemoproteus parasites, which might be used in a broad range of parasitology and genetic studies. Culicoides nubeculosus is an excellent experimental vector of subgenus Parahaemoproteus and is recommended for various experimental studies aiming investigation of sporogony of these pathogens.

Background Malaria remains a significant public health challenge in regions of the world where it is endemic. An unprecedented decline in malaria incidences was recorded during the last decade due to the availability of effective control interventions, such as the deployment of artemisinin-based combination therapy and insecticide-treated nets. However, according to the World Health Organization, malaria is staging a comeback, in part due to the development of drug resistance. Therefore, there is an urgent need to discover new anti-malarial drugs. This article reviews the literature on natural products with antiplasmodial activity that was reported between 2010 and 2017. Methods Relevant literature was sourced by searching the major scientific databases, including Web of Science, ScienceDirect, Scopus, SciFinder, Pubmed, and Google Scholar, using appropriate keyword combinations. Results and Discussion A total of 1524 compounds from 397 relevant references, assayed against at least one strain of Plasmodium, were reported in the period under review. Out of these, 39% were described as new natural products, and 29% of the compounds had IC50 ≤ 3.0 µM against at least one strain of Plasmodium. Several of these compounds have the potential to be developed into viable anti-malarial drugs. Also, some of these compounds could play a role in malaria eradication by targeting gametocytes. However, the research into natural products with potential for blocking the transmission of malaria is still in its infancy stage and needs to be vigorously pursued.

Background Seasonal malaria chemoprevention (SMC) consists of administration of sulfadoxine-pyrimethamine (SP) + amodiaquine (AQ) at monthly intervals to children during the malaria transmission period. Whether the addition of azithromycin (AZ) to SMC could potentiate the benefit of the intervention was tested through a double-blind, randomized, placebo-controlled trial. The effect of SMC and the addition of AZ, on malaria transmission and on the life history traits of Anopheles gambiae mosquitoes have been investigated. Methods The study included 438 children randomly selected from among participants in the SMC + AZ trial and 198 children from the same area who did not receive chemoprevention. For each participant in the SMC + AZ trial, blood was collected 14 to 21 days post treatment, examined for the presence of malaria sexual and asexual stages and provided as a blood meal to An. gambiae females using a direct membrane-feeding assay. Results The SMC treatment, with or without AZ, significantly reduced the prevalence of asexual Plasmodium falciparum (LRT X22 = 69, P 22 = 54, P 22 = 61, P X22 = 22.8, P 21 = 5.2, P = 0.02), suggesting a significant effect of AZ on gametocyte infectivity. There was a slight negative effect of SPAQ and SPAQ + AZ on mosquito survival compared to mosquitoes fed with blood from control children (LRTX22 = 330, P Conclusion This study demonstrates that SMC may contribute to a reduction in human to mosquito transmission of P. falciparum, and the reduced mosquito longevity observed for females fed on treated blood may increase the benefit of this intervention in control of malaria. The addition of AZ to SPAQ in SMC appeared to enhance the infectivity of gametocytes providing further evidence that this combination is not an appropriate intervention.

During the symptomatic human blood phase, malaria parasites replicate within red blood cells. Parasite proliferation relies on the uptake of nutrients, such as amino acids, from the host cell and blood plasma, requiring transport across multiple membranes. Amino acids are delivered to the parasite through the parasite-surrounding vacuolar compartment by specialized nutrient-permeable channels of the erythrocyte membrane and the parasitophorous vacuole membrane (PVM). However, further transport of amino acids across the parasite plasma membrane (PPM) is currently not well characterized. In this study, we focused on a family of Apicomplexan amino acid transporters (ApiATs) that comprises five members in Plasmodium falciparum. First, we localized four of the P. falciparum ApiATs (PfApiATs) at the PPM using endogenous green fluorescent protein (GFP) tagging. Next, we applied reverse genetic approaches to probe into their essentiality during asexual replication and gametocytogenesis. Upon inducible knockdown and targeted gene disruption, a reduced asexual parasite proliferation was detected for PfApiAT2 and PfApiAT4. Functional inactivation of individual PfApiATs targeted in this study had no effect on gametocyte development. Our data suggest that individual PfApiATs are partially redundant during asexual in vitro proliferation and fully redundant during gametocytogenesis of P. falciparum parasites. IMPORTANCE Malaria parasites live and multiply inside cells. To facilitate their extremely fast intracellular proliferation, they hijack and transform their host cells. This also requires the active uptake of nutrients, such as amino acids, from the host cell and the surrounding environment through various membranes that are the consequence of the parasite’s intracellular lifestyle. In this paper, we focus on a family of putative amino acid transporters termed ApiAT. We show expression and localization of four transporters in the parasite plasma membrane of Plasmodium falciparum-infected erythrocytes that represent one interface of the pathogen to its host cell. We probed into the impact of functional inactivation of individual transporters on parasite growth in asexual and sexual blood stages of P. falciparum and reveal that only two of them show a modest but significant reduction in parasite proliferation but no impact on gametocytogenesis, pointing toward dispensability within this transporter family. CA extern

The use of quantitative qRT-PCR assays for detection and quantification of late gametocyte stages has revealed the high transmission capacity of the human malaria parasite, Plasmodium falciparum. To understand how the parasite adjusts its transmission in response to in-host environmental conditions including antimalarials requires simultaneous quantification of early and late gametocytes. Here, we describe qRT-PCR assays that specifically detect and quantify early-stage P. falciparum gametocytes. The assays are based on expression of known early and late gametocyte genes and were developed using purified stage II and stage V gametocytes and tested in natural and controlled human infections. Genes pfpeg4 and pfg27 are specifically expressed at significant levels in early gametocytes with a limit of quantification of 190 and 390 gametocytes/mL, respectively. In infected volunteers, transcripts of pfpeg4 and pfg27 were detected shortly after the onset of blood stage infection. In natural infections, both early (pfpeg4/pfg27) and late gametocyte transcripts (pfs25) were detected in 71.2% of individuals, only early gametocyte transcripts in 12.6%, and only late gametocyte transcripts in 15.2%. The pfpeg4/pfg27 qRT-PCR assays are sensitive and specific for quantification of circulating sexually committed ring stages/early gametocytes and can be used to increase our understanding of epidemiological processes that modulate P. falciparum transmission.

Background Further reductions in malaria incidence as more countries approach malaria elimination require the identification and treatment of asymptomatic individuals who carry mosquito-infective Plasmodium gametocytes that are responsible for furthering malaria transmission. Assessing the relationship between total parasitaemia and gametocytaemia in field surveys can provide insight as to whether detection of low-density, asymptomatic Plasmodium falciparum infections with sensitive molecular methods can adequately detect the majority of infected individuals who are potentially capable of onward transmission. Methods In a cross-sectional survey of 1354 healthy children and adults in three communities in western Kenya across a gradient of malaria transmission (Ajigo, Webuye, and Kapsisywa–Kipsamoite), asymptomatic P. falciparum infections were screened by rapid diagnostic tests, blood smear, and quantitative PCR of dried blood spots targeting the varATS gene in genomic DNA. A multiplex quantitative reverse-transcriptase PCR assay targeting female and male gametocyte genes (pfs25, pfs230p), a gene with a transcriptional pattern restricted to asexual blood stages (piesp2), and human GAPDH was also developed to determine total parasite and gametocyte densities among parasitaemic individuals. Results The prevalence of varATS-detectable asymptomatic infections was greatest in Ajigo (42%), followed by Webuye (10%). Only two infections were detected in Kapsisywa. No infections were detected in Kipsamoite. Across all communities, children aged 11–15 years account for the greatest proportion total and sub-microscopic asymptomatic infections. In younger age groups, the majority of infections were detectable by microscopy, while 68% of asymptomatically infected adults (> 21 years old) had sub-microscopic parasitaemia. Piesp2-derived parasite densities correlated poorly with microscopy-determined parasite densities in patent infections relative to varATS-based detection. In general, both male and female gametocytaemia increased with increasing varATS-derived total parasitaemia. A substantial proportion (41.7%) of individuals with potential for onward transmission had qPCR-estimated parasite densities below the limit of microscopic detection, but above the detectable limit of varATS qPCR. Conclusions This assessment of parasitaemia and gametocytaemia in three communities with different transmission intensities revealed evidence of a substantial sub-patent infectious reservoir among asymptomatic carriers of P. falciparum. Experimental studies are needed to definitively determine whether the low-density infections in communities such as Ajigo and Webuye contribute significantly to malaria transmission.

Leucocytozoon sabrazesi is the intracellular protozoa of leucocytozoonosis, which is transmitted by the insect vectors and affects chickens in most subtropical and tropical regions of the globe, except South America, and causing enormous economic losses due to decreasing meat yield and egg production. In this study, L. sabrazesi gametocytes have been observed in the blood smears, and molecular methods have been used to analyse the occurrence and genetic diversity of L. sabrazesi in blood samples from 313 chickens raised in northern, western and southern parts of Thailand. The nested polymerase chain reaction (nested PCR) assay based on the cytb gene revealed that 80.51% (252/313) chickens were positive of L. sabrazesi. The phylogenetic analysis indicated that L. sabrazesi cytb gene is conserved in Thailand, showed 2 clades and 2 subclades with similarity ranged from 89.5 to 100%. The diversity analysis showed 13 and 18 haplotypes of the sequences from Thailand and from other countries, respectively. The entropy analyses of nucleic acid sequences showed 26 high entropy peaks with values ranging from 0.24493 to 1.21056, while those of amino acid sequences exhibited 5 high entropy peaks with values ranging from 0.39267 to 0.97012. The results; therefore, indicate a high molecular occurrence of L. sabrazesi in chicken blood samples with the associated factors that is statistically significant (p

Background Plasmodium malariae is the cause of the rare but severe form of malaria that sometimes affects individuals travelling to malaria-endemic regions. This report presents the unique case of a patient exhibiting severe malaria symptoms caused by P. malariae with no record of recent travel to any malaria-endemic areas. Case presentation An 81-year-old French woman was admitted to the emergency department with sustained fever and severe weakness for the past 5 days. She suffered from anaemia, thrombocytopenia, confusion, somnolence, pulmonary complications, and hypoxaemia. In the absence of any concrete aetiology that could explain the fever together with thrombocytopenia, physicians suspected malaria as a probable diagnosis. The LAMP-PCR and lateral flow test confirmed the presence of malaria parasite, Plasmodium sp. Microscopic examination (May-Grünwald Giemsa-stained thin blood smear) revealed the presence of trophozoites, schizonts, and gametocytes with 0.93 % parasitaemia. Conventional PCR amplification targeting 510 bp DNA fragment of small subunit ribosomal RNA (ssrRNA) and bidirectional sequencing identified the parasite as Plasmodium malariae. The travel history of this patient revealed her visits to several countries in Europe (Greece), North Africa (Tunisia and Morocco), and the West Indies (Dominican Republic). Of these, the latter was the only country known to be endemic for malaria at the time (three malaria parasite species were prevalent: Plasmodium falciparum, Plasmodium vivax, and P. malariae). The patient had most likely got infected when she visited the Dominican Republic in the summer of 2002. This time interval between the initial parasite infection (2002) till the onset of symptoms and its subsequent diagnosis (2020) is a reminder of the ability of P. malariae to persist in the human host for many years. Conclusions This report highlights the persistent nature and ability of P. malariae to cause severe infection in the host even after a prolonged time interval.

International audience; Plasmodium falciparum gametocytes modify the mechanical properties of their erythrocyte host to persist for several weeks in the blood circulation and to be available for mosquitoes. These changes are tightly regulated by the plasmodial phosphodiesterase delta that decreases both the stiffness and the permeability of the infected host cell. Here, we address the effect of the phosphodiesterase inhibitor tadalafil on deformability and permeability of gametocyteinfected erythrocytes. We show that this inhibitor drastically increases isosmotic lysis of gametocyte-infected erythrocytes and impairs their ability to circulate in an in vitro model for splenic retention. These findings indicate that tadalafil represents a novel drug lead potentially capable of blocking malaria parasite transmission by impacting gametocyte circulation.

Highlights • Sexual reproduction is obligate for malaria parasite transmission. • Parasites have evolved “strategies” to maximise the success of sexual reproduction. • Evolutionary and ecological theories can explain parasite reproductive strategies. • Parasite strategies could undermine transmission-blocking interventions. • Targeting parasite strategies offers novel opportunities for interventions., Malaria parasites exhibit a complex lifecycle, requiring extensive asexual replication in the liver and blood of the vertebrate host, and in the haemocoel of the insect vector. Yet, they must also undergo a single round of sexual reproduction, which occurs in the vector’s midgut upon uptake of a blood meal. Sexual reproduction is obligate for infection of the vector and thus, is essential for onwards transmission to new hosts. Sex in malaria parasites involves several bottlenecks in parasite number, making the stages involved attractive targets for blocking disease transmission. Malaria parasites have evolved a suite of adaptations (“strategies”) to maximise the success of sexual reproduction and transmission, which could undermine transmission-blocking interventions. Yet, understanding parasite strategies may also reveal novel opportunities for such interventions. Here, we outline how evolutionary and ecological theories, developed to explain reproductive strategies in multicellular taxa, can be applied to explain two reproductive strategies (conversion rate and sex ratio) expressed by malaria parasites within the vertebrate host.

Background Malaria is a life-threatening, multisystem disease caused by the plasmodial parasite with a global incidence of approximately 229 million annually. The parasites are known to have unique and crucial interactions with various body tissues during its life cycle, notably the liver, spleen, and recent work has shown the bone marrow to be a reservoir of infection. Methods This study is a case series of patients in whom examination of bone marrow revealed malarial parasites. A retrospective record review of 35 parasite-positive bone marrow specimens examined at Aga Khan University Hospital (AKUH), Karachi, Pakistan, over the years 2007 to 2015 was conducted. Bone marrow aspirates were collected as per International Council for Standardization in Haematology (ICSH) guidelines. Results The median age of patients was 22 years (range 1–75), and 60 % (n = 21) were male. 22 patients had evidence of Plasmodium falciparum, 12 had evidence of Plasmodium vivax and 1 patient had a mixed infection. Gametocytes and trophozoites were the most common stages identified on both peripheral blood and bone marrow examinations. Indications for bone marrow examination included fever of unknown origin and the workup of cytopenias and malignancies. Conclusions The incidental finding of Plasmodium in samples of bone marrow suggests the reticuloendothelial system may be regularly harbour these parasites, be the infection acute or chronic in character.

Background Coccidiosis caused by Eimeria stiedae is a widespread and economically significant disease of rabbits. The lack of studies on the life-cycle development and host interactions of E. stiedae at the molecular level has hampered our understanding of its pathogenesis. Methods In this study, we present a comprehensive transcriptome landscape of E. stiedae to illustrate its dynamic development from unsporulated oocysts to sporulated oocysts, merozoites, and gametocytes, and to identify genes related to parasite-host interactions during parasitism using combined PacBio single-molecule real-time and Illumina RNA sequencing followed by bioinformatics analysis and qRT-PCR validation. Results In total, 12,582 non-redundant full-length transcripts were generated with an average length of 1808 bp from the life-cycle stages of E. stiedae. Pairwise comparisons between stages revealed 8775 differentially expressed genes (DEGs) showing highly significant description changes, which compiled a snapshot of the mechanisms underlining asexual and sexual biology of E. stiedae including oocyst sporulation between unsporulated and sporulated oocysts; merozoite replication between sporulated oocysts and merozoites; and gametophyte development and gamete generation between merozoites and gametocytes. Further, 248 DEGs were grouped into nine series clusters and five groups by expression patterns, and showed that parasite–host interaction-related genes predominated in merozoites and gametocytes and were mostly involved in steroid biosynthesis and lipid metabolism and carboxylic acid. Additionally, co-expression analyses identified genes associated with development and host invasion in unsporulated and sporulated oocysts and immune interactions during gametocyte parasitism. Conclusions This is the first study, to our knowledge, to use the global transcriptome profiles to decipher molecular changes across the E. stiedae life cycle, and these results not only provide important information for the molecular characterization of E. stiedae, but also offer valuable resources to study other apicomplexan parasites with veterinary and public significance. Graphical Abstract

Background Plasmodium knowlesi is now the major cause of human malaria in Malaysia, complicating malaria control efforts that must attend to the elimination of multiple Plasmodium species. Recent advances in the cultivation of P. knowlesi erythrocytic-stage parasites in vitro, transformation with exogenous DNA, and infection of mosquitoes with gametocytes from culture have opened up studies of this pathogen without the need for resource-intensive and costly non-human primate (NHP) models. For further understanding and development of methods for parasite transformation in malaria research, this study examined the activity of various trans-species transcriptional control sequences and the influence of Plasmodium vivax centromeric (pvcen) repeats in plasmid-transfected P. knowlesi parasites. Methods In vitro cultivated P. knowlesi parasites were transfected with plasmid constructs that incorporated Plasmodium vivax or Plasmodium falciparum 5′ UTRs driving the expression of bioluminescence markers (firefly luciferase or Nanoluc). Promoter activities were assessed by bioluminescence, and parasites transformed with human resistant allele dihydrofolate reductase-expressing plasmids were selected using antifolates. The stability of transformants carrying pvcen-stabilized episomes was assessed by bioluminescence over a complete parasite life cycle through a rhesus macaque monkey, mosquitoes, and a second rhesus monkey. Results Luciferase expression assessments show that certain P. vivax promoter regions, not functional in the more evolutionarily-distant P. falciparum, can drive transgene expression in P. knowlesi. Further, pvcen repeats may improve the stability of episomal plasmids in P. knowlesi and support detection of NanoLuc-expressing elements over the full parasite life cycle from rhesus macaque monkeys to Anopheles dirus mosquitoes and back again to monkeys. In assays of drug responses to chloroquine, G418 and WR9910, anti-malarial half-inhibitory concentration (IC50) values of blood stages measured by NanoLuc activity proved comparable to IC50 values measured by the standard SYBR Green method. Conclusion All three P. vivax promoters tested in this study functioned in P. knowlesi, whereas two of the three were inactive in P. falciparum. NanoLuc-expressing, centromere-stabilized plasmids may support high-throughput screenings of P. knowlesi for new anti-malarial agents, including compounds that can block the development of mosquito- and/or liver-stage parasites.

Background Poor knowledge on the afebrile Plasmodium falciparum biology limits elimination approaches to target asymptomatic malaria. Therefore, the association of parasite factors involved in cytoadhesion, parasite multiplication and gametocyte maturation with afebrile malaria was assessed. Methods Plasmodium falciparum isolates were collected from febrile (axillary temperature ≥ 37.5 °C or a reported fever in the previous 24 h) and afebrile (fever neither at the visit nor in the previous 24 h) individuals residing in Southern Mozambique. var, PfSir2a and Pfs25 transcript levels were determined by reverse transcriptase quantitative PCRs (RT-qPCRs) and compared among 61 pairs of isolates matched by parasite density, age and year of sample collection. Results The level of varC and PfSir2a transcripts was higher in P. falciparum isolates from afebrile individuals (P ≤ 0.006), while varB and DC8 genes (P ≤ 0.002) were higher in isolates from individuals with febrile infections. After adjusting the analysis by area of residence, doubling the relative transcript unit (RTU) of varC and PfSir2a was associated with a 29.7 (95% CI 4.6–192.3) and 8.5 (95% CI 1.9–32.2) fold increases, respectively, of the odds of being afebrile. In contrast, doubling the RTU of varB and DC8 was associated with a 0.8 (95% CI 0.05–0.6) and 0.2 (95% CI 0.04–0.6) fold changes, respectively, of the odds of being afebrile. No significant differences were found for Pfs25 transcript levels in P. falciparum isolates from afebrile and febrile individuals. Conclusions var and gametocyte-specific transcript patterns in febrile and afebrile infections from southern Mozambique matched by age, parasite density and recruitment period suggest similar transmissibility but differential expression of variant antigens involved in cytoadhesion and immune-evasion.

Background Natural exposure to gametocytes can result in the development of immunity against the gametocyte by the host as well as genetic diversity in the gametocyte. This study evaluated the quantity and quality of natural immune responses against a gametocyte antigen, Pfs230 as well as the prevalence and diversity of gametocytes circulating in children living in two communities in southern Ghana. Methods Whole blood (2.5 ml) was collected from 137 non-febrile school children aged between 6 and 12 years old quarterly for a 6-month period. A drop of blood was used to prepare thick and thin blood films for parasite prevalence and density estimation. Subsequently, stored plasma samples were used in ELISAs assays to measure antibody responses and avidity against Pfs230. RNA was extraction from Trizol preserved packed cells and subsequently converted to complementary DNA (cDNA) which was used for reverse transcriptase PCR (RT-PCR) to determine gametocytes prevalence and diversity. Results Gametocyte carriage in the peak season (July) determined by Pfg377 RT-PCR was 49.2% in Obom and 22.2% in Abura, and was higher than that determined by microscopy. Gametocyte diversity was low and predominated by the same allele at both sites. The relative avidity index for antibodies measured in Abura was higher than that recorded in Obom at all time points although Pfs230 IgG concentrations were significantly high (P

Avian communities from South America harbor an extraordinary diversity of Leucocytozoon species (Haemosporida, Leucocytozoidae). Here, of 890 birds sampled, 10 (1.2%) were infected with Leucocytozoon parasites. Among them, two new species were discovered and described. Leucocytozoon grallariae sp. nov. and Leucocytozoon neotropicalis sp. nov. were found in non-migratory highland passeriforms belonging to the Grallaridae and Cotingidae, respectively. They both possess gametocytes in fusiform host cells. However, due to combining microscopic examination and molecular detection, it was revealed that these parasites were present in co-infections with other Leucocytozoon species, which gametocytes develop in roundish host cells, therefore exhibiting two highly distant parasite lineages isolated from the same samples. Remarkably, the lineages obtained by cloning the mtDNA genomes were not captured by the classic nested PCR, which amplifies a short fragment of cytochrome b gene. Phylogenetic analyses revealed that the lineages obtained by the classic nested PCR clustered with parasites possessing gametocytes in roundish host cells, while the lineages obtained by the mtDNA genome PCR protocol were closely related to Leucocytozoon parasites possessing gametocytes in fusiform host cells. These findings suggest problems with the sensitivity of the molecular protocols commonly used to detect Leucocytozoon species. A detailed analysis of the primers used in the classic nested PCR revealed a match with DNA sequences from those parasites that possess gametocytes in roundish host cells (i.e., Leucocytozoon fringillinarum), while they differ with the orthologous regions in the mtDNA genomes isolated from the samples containing the two new species. Since these are mixed infections, none of the lineages detected in this study can be assigned accurately to the new Leucocytozoon morphospecies that develops in fusiform host cells. However, phylogenetic analyses allowed us to hypothesize their most probable associations. This study highlights the need for developing detection methods to assess the diversity of Leucocytozoon parasites accurately., Graphical abstract Image 1, Highlights • Molecular diversity of Leucocytozoon is underestimated. • We described two new Leucocytozoon species infecting passerines endemic of Neotropics. • Commonly PCR protocols failed to detect Leucocytozoon lineages from Neotropical region.

Plasmodium parasites can infect great variety of bird species around the world inflicting the so called avian malaria, an illness that could be fatal in some cases and consequently, should be monitored and widely included into conservation programs. The aim of this study was to characterize two lineages of Plasmodium (Haemamoeba) lutzi found in some birds in the Atlantic Forest of Minas Gerais - Brazil, that were morphologically identified after blood smears analyses under light microscopy and molecularly by sequencing the mitochondrial cytochrome b gene (cyt b). Besides these two lineages could be clearly morphologically identified as P.(H.) lutzi, some variations in comparison with its original description were noticed: absence of meronts and gametocytes (early and fully grown) in polychromatic erythrocytes, the larger size of pigment granules in meronts and gametocytes, and the presence of small vacuoles between pigment accumulation in fully grow macrogametocytes. Moreover, a certain degree of genetic intraspecific diversity was also observed across the lineages of P. (H.) lutzi, indicating the existence of polymorphisms within this taxon, which is uncommon in Haemosporida. These results allow discussion about species boundaries within avian hemosporidians and highlight the importance of multidisciplinary approaches for a more efficient species identification and characterization.

The deadliest form of human malaria is primarily caused by the protozoan parasite Plasmodium falciparum. These parasites establish pathogenicity in the human host with a very low number of sexual forms or gametocytes, which are transmitted to the mosquitoes. Several studies have reported that exposing artemisinin-sensitive P. falciparum rings to a low concentration of dihydroartemisinin (DHA) results in dormancy, and the artemisinin-induced dormant (AID) forms are recovered into normal growth stages after 5–20 days. In this study, artemisinin-resistant P. falciparum parasites were tested for the development of AID forms and their recovery. Interestingly, it was found that exposure of an asynchronous culture of artemisinin-resistant P. falciparum IPC 5202 to DHA, a line carrying a mutation in the PfK13 gene that is linked to artemisinin resistance, also results in dormancy. Both the ring and some late stages of these AID forms recovered after 10–15 days. Furthermore, a high proportion of the recovered dormant forms developed into sexual forms or gametocytes after 3–4 weeks, which is almost a 7–8 times higher rate of conversion of asexual to sexual forms (gametocytes) or the malaria transmissible forms. In contrast, only early ring forms of artemisinin-sensitive parasites recovered slowly, and additional exposure of these parasites to artemisinin resulted in complete clearance within a week. This is in contrast to the resistant parasites exposed to a second dose of artemisinin, which resulted in a very high rate of dormancy and recovery into sexual forms or gametocytes.

BACKGROUND: The Belgian Reference Laboratory for Plasmodium offers a free-of-charge reference testing of malaria-positive or doubtful samples to clinical laboratories. METHODS: The final malaria diagnosis from the Reference Laboratory (microscopy, rapid diagnostic tests (RDTs) and Plasmodium species-specific PCR) were compared with the final diagnosis from peripheral Belgian laboratories. The Reference Laboratory reports were analysed for all samples submitted between 2013 and 2017. Criteria assessed included the diagnosis of malaria, Plasmodium species identification including mixed infections, and in case of Plasmodium falciparum, the parasite density and the presence of sexual and asexual stages. RESULTS: A total of 947 non-duplicate samples were included. Reference testing confirmed 96.3% (893/927) and 90.0% (18/20) samples submitted as positive and negative, respectively, the two missed diagnoses were samples with Plasmodium ovale and Plasmodium malariae. Submitting laboratories had correctly identified P. falciparum in 95.1% (508/534) samples with P. falciparum single infection. They had correctly diagnosed the species in 62.9% (95/151) single non-falciparum samples and had reported 'non-falciparum' in another 26 (17.2%) samples; most errors occurred among P. malariae (n = 8/21, 38.1%) and P. ovale (n = 14/51, 27.5%). Only one of the 21 mixed Plasmodium species infections had been diagnosed as such by the submitting laboratories; in three of them, P. falciparum had been overlooked. Taken single and mixed infections together, P. falciparum was diagnosed in 98.6% (546/554) samples. Among 471 single P. falciparum samples available for comparison, laboratories had correctly reported parasite densities above 2% in 87.5% (70/80) samples; they had incorrectly reported parasite densities > 2% in an extra 52 (8.9%) samples. Laboratories had correctly reported P. falciparum schizonts and gametocytes in 25.6% (11/43) and 56.7% (17/30) samples, respectively. CONCLUSION: Diagnostic laboratories in a malaria non-endemic setting provided excellent diagnosis of malaria and P. falciparum, reasonably good diagnosis of non-falciparum infections and acceptable calculation of P. falciparum parasite density. ispartof: MALARIA JOURNAL vol:18 issue:1 ispartof: location:England status: published

Background Optimal adoption of the malaria transmission-blocking strategy is currently limited by lack of safe and efficacious drugs. This has sparked the exploration of different sources of drugs in search of transmission-blocking agents. While plant species have been extensively investigated in search of malaria chemotherapeutic agents, comparatively less effort has been channelled towards exploring them in search of transmission-blocking drugs. Artemisia afra (Asteraceae), a prominent feature of South African folk medicine, is used for the treatment of a number of diseases, including malaria. In search of transmission-blocking compounds aimed against Plasmodium parasites, the current study endeavoured to isolate and identify gametocytocidal compounds from A. afra. Methods A bioassay-guided isolation approach was adopted wherein a combination of solvent–solvent partitioning and gravity column chromatography was used. Collected fractions were continuously screened in vitro for their ability to inhibit the viability of primarily late-stage gametocytes of Plasmodium falciparum (NF54 strain), using a parasite lactate dehydrogenase assay. Chemical structures of isolated compounds were elucidated using UPLC-MS/MS and NMR data analysis. Results Two guaianolide sesquiterpene lactones, 1α,4α-dihydroxybishopsolicepolide and yomogiartemin, were isolated and shown to be active (IC50

Rabbit coccidiosis is a common parasitic disease and responsible for enormous economic losses in the rabbit industry. Eimeria intestinalis, one of the highly pathogenic and common Eimeria species infecting rabbits, is considered as an indispensable species for the development of live oocyst vaccines against rabbit coccidiosis. In this study, we report the successful selection of a precocious line (EIP8) from a wild-type strain of E. intestinalis (WT) by successively collecting and propagating the early excreted progeny oocysts. The EIP8 line had a prepatent period of only 132 h compared to 204 h for the WT. Oocysts of EIP8 were notably different from those produced by the WT strain by their significantly larger size (mean length: 29.3 vs 27.6 μm and mean width 20.5 vs 19.8 μm). Examination of tissue sections prepared from EIP8-infected rabbits revealed that this precocious line undergoes only two generations of schizogony before differentiating into gametocytes by 120 h post-infection. In contrast, WT parasites undergo three generations of schizogony and gametocytes are present by 168 h post-infection. EIP8 multiplication capacity reduced by more than 35-fold and a concomitant decrease in pathogenicity was detected. Interestingly, immunization with 103 or 104 EIP8 oocysts provided sufficient protection against homologous challenge with wild-type parasites, as body weight gain of immunized and challenged rabbits was similar to that of untreated animals, as well as more than 90% reduction of oocyst output was detected in immunized and challenged animals when compared to unimmunized and challenged animals. Together, these results show that the EIP8 precocious line of E. intestinalis is an attenuated immunogenic strain and a suitable candidate for the development of a live vaccine against rabbit coccidiosis.

Gametocyte proteins are being explored as potential vaccine candidates against Eimeria sp. in chicken since they are the components of the resilient oocyst wall. The aim of this study was to investigate the immunoprophylactic efficacy of recombinant Eimeria tenella gametocyte antigen 22 (EtGam22) in chickens against homologous oocyst challenge. Broiler chicks were subcutaneously immunized individually with 100 μg of recombinant EtGam22 adjuvanted with Montanide ISA 71 VG at 7 days of age and boosted 2 weeks later. The immunized chickens were challenged individually with 1 × 104 sporulated oocysts of E. tenella 1 week post-booster immunization. The anti-EtGam22 IgY and serum cytokine response was measured post-immunization. The results showed that the anti-EtGam22 IgY antibody, serum IFN-γ, IL-2, TGF-β, and IL-4 levels in chickens vaccinated with recombinant protein were significantly increased post-immunization as compared to unimmunized challenged controls (P

Background The development of malaria transmission-blocking strategies including the generation of malaria refractory mosquitoes to replace the wild populations through means of gene drives hold great promise. The standard membrane feeding assay (SMFA) that involves mosquito feeding on parasitized blood through an artificial membrane system is a vital tool for evaluating the efficacy of transmission-blocking interventions. However, despite the availability of several published protocols, the SMFA remains highly variable and broadly insensitive. Methods The SMFA protocol was optimized through coordinated culturing of Anopheles coluzzii mosquitoes and Plasmodium falciparum parasite coupled with placing mosquitoes under a strict dark regime before, during, and after the gametocyte feed. Results A detailed description of essential steps is provided toward synchronized generation of highly fit An. coluzzii mosquitoes and P. falciparum gametocytes in preparation for an SMFA. A dark-infection regime that emulates the natural vector-parasite interaction system is described, which results in a significant increase in the infection intensity and prevalence. Using this optimal SMFA pipeline, a series of putative transmission-blocking antimicrobial peptides (AMPs) were screened, confirming that melittin and magainin can interfere with P. falciparum development in the vector. Conclusion A robust SMFA protocol that enhances the evaluation of interventions targeting human malaria transmission in laboratory setting is reported. Melittin and magainin are identified as highly potent antiparasitic AMPs that can be used for the generation of refractory Anopheles gambiae mosquitoes.

Background & objectives: Malaria has remained a global health problem despite the effective control and treatment measures. In the backdrop of drug resistance, developing novel hybrid molecules targeting the sexual stages (gametocytes) of the human malaria parasite Plasmodium falciparum is of great significance. Recently, chalcone- based polyphenols have generated a great interest in the malaria research community worldwide due to their ease of synthesis and significant biological activity. The primary objective of this study was to investigate the interaction of a newly synthesized quinoline-appended chalcone derivative (ADMQ) with gametocyte specific proteins, Pfg 27 and Pfs 25 and explore its in vitro gametocytocidal potential. Methods: The characterization of ligand-protein interactions at the atomistic level was done by a simulation strategy that combines molecular docking and molecular dynamics (MD) simulation in a coherent workflow. The X-ray crystal structure of Pfg 27 was retrieved from protein data bank and Pfs 25 was built using the Iterative Threading ASSembly Refinement (I-TASSER) server. The detailed interaction of both ADMQ and a known gametocytocidal agent, methylene blue (MB) (used as a positive control) with gametocyte proteins Pfg 27 and Pfs 25 was studied with a 50 ns explicit MD simulation. The ligand binding pose in terms of glide score, molecular mechanics-generalized born surface area (MM-GBSA) binding energies, protein-ligand root-mean-square-deviation (RMSD) and secondary structure elements (SSE) changes were analyzed accordingly. The direct effect of ADMQ on structural integrity of P. falciparum gametocytes was also examined using in vitro microscopy. Results: The analogous Glide score and MM-GBSA free energy of binding indicated stable interactions for both ADMQ and MB harboured in the active site of targeted gametocyte proteins, Pfg 27 and Pfs 25, separately. Explicit MD simulation by Desmond software package indicated similar distinguishable conformational changes in the active site of target polypeptide chain due to the specific accommodation of ADMQ molecule. The simulation also manifested comparable mechanistic profile in terms of protein-ligand RMSD and changes in secondary structure elements (SSE). Further, ADMQ treatment was found to adversely affect the structural integrity of gametocytes, which resulted in appearance of vesicles protruding from the gametocytes. Interpretation & conclusion: The consolidated in silico molecular modeling and in vitro study described herein may give an insight into the interaction patterns of quinoline-chalcone hybrids with critical gametocyte proteins in the mosquito. This study will possibly pave the way for further exploration of similar heterocyclic quinoline-chalcone hybrids to open up new avenues in drug candidate development against P. falciparum gametocytes.

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.

Background The ability to culture Plasmodium falciparum continuously in vitro has enabled stable access to asexual and sexual parasites for malaria research. The portfolio of isolates has remained limited and research is still largely based on NF54 and its derived clone 3D7. Since 1978, isolates were collected and cryopreserved at Radboudumc from patients presenting at the hospital. Here, procedures are described for culture adaptation of asexual parasites, cloning and production of sexual stage parasites responsible for transmission (gametocytes) and production of oocysts in Anopheles mosquitoes. This study aimed to identify new culture-adapted transmissible P. falciparum isolates, originating from distinct geographical locations. Methods Out of a collection of 121 P. falciparum isolates stored in liquid nitrogen, 21 from different geographical origin were selected for initial testing. Isolates were evaluated for their ability to be asexually cultured in vitro, their gametocyte production capacity, and consistent generation of oocysts. Results Out of 21 isolates tested, twelve were excluded from further analysis due to lack of mature gametocyte production (n = 1) or generation of satisfactory numbers of oocysts in mosquitoes (n = 11). Nine isolates fulfilled selection criteria and were cloned by limiting dilution and retested. After cloning, one isolate was excluded for not showing transmission. The remaining eight isolates transmitted to Anopheles stephensi or Anopheles coluzzii mosquitoes and were categorized into two groups with a reproducible mean oocyst infection intensity above (n = 5) or below five (n = 3). Conclusions These new P. falciparum culture-adapted isolates with reproducible transmission to Anopheles mosquitoes are a valuable addition to the malaria research tool box. They can aid in the development of malaria interventions and will be particularly useful for those studying malaria transmission.

Background Efforts to study the biology of Plasmodium vivax liver stages, particularly the latent hypnozoites, have been hampered by the limited availability of P. vivax sporozoites. Anopheles stephensi is a major urban malaria vector in Goa and elsewhere in South Asia. Using P. vivax patient blood samples, a series of standard membrane-feeding experiments were performed with An. stephensi under the US NIH International Center of Excellence for Malaria Research (ICEMR) for Malaria Evolution in South Asia (MESA). The goal was to understand the dynamics of parasite development in mosquitoes as well as the production of P. vivax sporozoites. To obtain a robust supply of P. vivax sporozoites, mosquito-rearing and mosquito membrane-feeding techniques were optimized, which are described here. Methods Membrane-feeding experiments were conducted using both wild and laboratory-colonized An. stephensi mosquitoes and patient-derived P. vivax collected at the Goa Medical College and Hospital. Parasite development to midgut oocysts and salivary gland sporozoites was assessed on days 7 and 14 post-feeding, respectively. The optimal conditions for mosquito rearing and feeding were evaluated to produce high-quality mosquitoes and to yield a high sporozoite rate, respectively. Results Laboratory-colonized mosquitoes could be starved for a shorter time before successful blood feeding compared with wild-caught mosquitoes. Optimizing the mosquito-rearing methods significantly increased mosquito survival. For mosquito feeding, replacing patient plasma with naïve serum increased sporozoite production > two-fold. With these changes, the sporozoite infection rate was high (> 85%) and resulted in an average of ~ 22,000 sporozoites per mosquito. Some mosquitoes reached up to 73,000 sporozoites. Sporozoite production could not be predicted from gametocyte density but could be predicted by measuring oocyst infection and oocyst load. Conclusions Optimized conditions for the production of high-quality P. vivax sporozoite-infected An. stephensi were established at a field site in South West India. This report describes techniques for producing a ready resource of P. vivax sporozoites. The improved protocols can help in future research on the biology of P. vivax liver stages, including hypnozoites, in India, as well as the development of anti-relapse interventions for vivax malaria.

Background A goal of malaria epidemiological interventions is the detection and treatment of parasite reservoirs in endemic areas—an activity that is expected to reduce local transmission. Since the gametocyte is the only transmissible stage from human host to mosquito vector, this study evaluated the pre and post presence of gametocytes during a mass screening and treatment (MST) intervention conducted during 2013 in East Nusa Tenggara, Indonesia. Methods RT-qPCR targeting pfs25 and pvs25 transcripts—gametocyte molecular markers for Plasmodium falciparum and Plasmodium vivax, respectively, was performed to detect and quantify gametocytes in blood samples of P. falciparum and P. vivax-infected subjects over the course of the MST study. The presence of both asexual and sexual parasites in microscopic and submicroscopic infections was compared from the start and end of the MST, using proportion tests as well as parametric and non-parametric tests. Results Parasite prevalence remained unchanged for P. falciparum (6% = 52/811 versus 7% = 50/740, p = 0.838), and decreased slightly for P. vivax (24% = 192/811 versus 19% = 142/740, p = 0.035) between the MST baseline and endpoint. No significant difference was observed in gametocyte prevalence for either P. falciparum (2% = 19/803 versus 3% = 23/729, p = 0.353, OR = 1.34, 95%CI = 0.69–2.63), or P. vivax (7% = 49/744 versus 5% = 39/704, p = 0.442, OR = 0.83, 95%CI = 0.52–1.31). Even though there was an insignificant difference between the two time points, the majority of parasite positive subjects at the endpoint had been negative at baseline (P. falciparum: 66% = 29/44, P. vivax: 60% = 80/134). This was similarly demonstrated for the transmissible stage—where the majority of gametocyte positive subjects at the endpoint were negative at baseline (P. falciparum: 95% = 20/21, P. vivax: 94% = 30/32). These results were independent of treatment provided during MST activities. No difference was demonstrated in parasite and gametocyte density between both time points either in P. falciparum or P. vivax. Conclusion In this study area, similar prevalence rates of P. falciparum and P. vivax parasites and gametocytes before and after MST, although in different individuals, points to a negligible impact on the parasite reservoir. Treatment administration based on parasite positivity as implemented in the MST should be reevaluated for the elimination strategy in the community. Trial registration Clinical trials registration NCT01878357. Registered 14 June 2013, https://www.clinicaltrials.gov/ct2/show/NCT01878357.

As malaria elimination becomes a possibility the focus of interventions changes from vector control to disease control. It is important that treatment occurs early during an infection in order for it to be efficacious, especially at the population level. The time between the onset of symptoms and treatment seeking is, therefore, crucial. Following a census and an oral autopsy survey of the inhabitants of Furvela, a village in southern Mozambique, a malaria post (MP) where malaria was diagnosed and treated was established in 2001. The time between the onset of symptoms and attendance at the MP was determined and compared to the severity of disease. A cross-sectional survey was also conducted, in 2007, to determine prevalence amongst 235 children aged between 6 months and 15 years of age. Malaria was hyperendemic in the village and was responsible for most deaths reported from the two years prior to the start of the project. In the prevalence survey 74% of two-to-four-year-old children had malaria parasites. The likelihood of being parasite positive was significantly higher in children living in houses with roofs made of traditional materials compared to those living in houses with tin roofs. At the start of the project only 12% of residents owned or used a mosquito net, most of which were not treated with insecticide. However, even before any formal intervention, malaria declined in the village between 2001 and 2007, but there was a rebound in later years. Nevertheless, the relative proportion of patients who had to be referred to the hospital declined significantly in the latter years of the project, and the incidence of both Plasmodium ovale and P. malariae also decreased significantly. Overall 16698 patients, the majority of which were under one year of age, attended the MP between 2001 and 2010. The proportion of patients with a positive slide for P. falciparum remained relatively constant throughout the study (mean 0.66 std. dev. 0.3) Most of the patients came from the village of Furvela, or its environs, but some came from the nearby town, ostensibly because of the good treatment they received. Infection rates increased up to the first three years of life to a peak incidence of 92% at 31 months. Children with fever had higher parasite densities than those without fever. Mothers generally bought their children to the MP on the second day of symptoms but on the first day if they had fever. Older patients, with lower density infections, delayed in coming for treatment. These patients may harbour sub-microscopic gametocytes which would help maintain transmission in the village. Mothers acted appropriately in their treatment seeking behaviour. The establishment of village-based MPs are an effective way of providing adequate diagnosis and treatment in villages such as Furvela.

BackgroundThe emergence of artemisinin resistance in Southeast Asia andPlasmodium falciparum kelch13propeller gene mutations in sub-Saharan African pose the greatest threat to global efforts to control malaria. This is a critical concern in Uganda, where artemisinin-based combination therapy (ACT) is the first-line treatment for uncomplicated falciparum. The objective of this study was to compare the efficacy and safety of dihydroartemisinin–piperaquine (DHA–PQ) and artemether–lumefantrine (AL) for the treatment of uncomplicated falciparum malaria in Ugandan children.MethodsA search of PubMed and the Cochrane Central Register of Controlled Trials for retrieving randomized controlled trials comparing the efficacy and safety of DHA–PQ and AL for treatment of uncomplicated falciparum malaria in Ugandan children was done. The search was performed up to 31 August 2020. The data extracted from eligible studies and pooled as risk ratio (RR) with a 95% confidence interval (CI), using Rev Man Software (5.4). The protocol was registered in PROSPERO, ID: CRD42020182354.ResultsEleven trials were included in this review and two of them only included under safety outcome. Total 3798 participants were enrolled. The PCR unadjusted treatment failure was significantly lower with DHA–PQ at day 28 (RR 0.30, 95% CI 0.19–0.49; participants = 7863; studies = 5; I2 = 93%, low quality evidence) and at day 42 (RR 0.53, 95% CI 0.38–0.76; participants = 1618; studies = 4; I2 = 79%, moderate quality of evidence). The PCR adjusted treatment failure at day 42 was significantly lower with DHA–PQ treatment group (RR 0.45, 95% CI 0.28 to 0.72; participants = 1370; studies = 5, high quality of evidence), and it was below 5% in both arms at day 28 (moderate quality of evidence). AL showed a longer prophylactic effect on new infections which may last for up to 63 days (PCR-adjusted treatment failure: RR 2.04, 95% CI 1.13–3.70; participants = 1311; studies = 2, moderate quality of evidence). Compared to AL, DHA–PQ was associated with a slightly higher frequency of cough (RR 1.07, 95% CI 1.01 to 1.13; 2575 participants; six studies; high quality of evidence). In both treatment groups, the risk of recurrent parasitaemia due to possible recrudescence was less than 5% at day 28. The appearance of gametocyte between 29 and 42 days was also significantly lower in DHA–PQ than AL (RR 0.26, 95% CI 0.12 to 0.56; participants = 623; studies = 2; I2 = 0%).ConclusionCompared to AL, DHA–PQ appeared to reduce treatment failure and gametocyte carriage in Ugandan children. This may trigger DHA–PQ to become the first-line treatment option. Both treatments were safe and well-tolerated.