Search

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

Background Plasmodium falciparum gametocytaemia has been associated with anaemia. The aim of this review was to synthesize available evidence on the comparative effect of dihydroartemisinin-piperaquine (DHA-PQ) and artemether-lumefantrine (AL) on gametocyte clearance and haemoglobin recovery in children with uncomplicated P. falciparum malaria in Africa. Methods A systematic literature search was undertaken to identify relevant articles from online databases. The search was performed from August 2020 to 30 April 2021. Extracted data from eligible studies were pooled as risk ratios with 95% confidence intervals (CI). Results Gametocyte carriage was reduced in both treatment groups, with no significant difference found between the groups. However, on days 28 and 42, a significant increase in serum haemoglobin level from baseline was observed in the DHA-PQ group (standardized mean difference 0.15, 95% CI 0.05–0.26; participants=2715; studies=4; I2=32%, high quality of evidence) compared with the AL group (mean difference 0.35, 95% CI 0.12–0.59; participants=1434; studies=3; I2=35%, high quality of evidence). Conclusion DHA-PQ had a greater impact on haemoglobin recovery than AL on days 28 and 42; this difference was significant.

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.

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.

Background Both capillary and venous blood samples have been interchangeably used for the diagnosis of malaria in Ethiopia. However, Plasmodium parasites are thought to be more concentrated in capillary than in venous blood. Hence, selecting a sample source where parasites are more concentrated is indispensable approach in order to maximize the accuracy of blood film microscopy. Therefore, the present study aimed to compare the detection rate and the parasitemia level of Plasmodium species from conventional capillary and venous blood films, and buffy coat preparations. Methods A facility based cross-sectional study was conducted from Feburary to March 2020 among 210 febrile patients attending Hamusite health center, northwest Ethiopia. Capillary and venous blood samples were collected and buffy coat was prepared from each sample. Thin and thick blood films were prepared, stained, and examined microscopically following standard protocol. Data were analysed using Statistical Package for Social Sciences Software version 20 and Med-Calc software version 19.3. Results Capillary blood buffy coat (61/210, 29.0%) had significantly higher detection rate as compared to capillary (48/210, 22.9%) and venous (42/210, 20.0%) blood films (p p = 0.070) in detecting Plasmodium species. The highest and the lowest mean asexual stage parasite counts were found in capillary blood buffy coat (4692.88) and venous blood (631.43) films, respectively showing significant variations (p Conclusion Capillary blood buffy coat samples showed the highest sensitivity in detecting and quantitating malaria parasites that its use should be promoted in clinical settings. However, conventional capillary and venous blood films could be used interchangeably.

A novel series of antimalarial benzimidazole derivatives incorporating phenolic Mannich base side chains at the C2 position, which possess dual asexual blood and sexual stage activities, is presented. Structure-activity relationship studies revealed that the 1-benzylbenzimidazole analogues possessed submicromolar asexual blood and sexual stage activities in contrast to the 1H-benzimidazole analogues, which were only active against asexual blood stage (ABS) parasites. Further, the former demonstrated microtubule inhibitory activity in ABS parasites but more significantly in stage II/III gametocytes. In addition to being bona fide inhibitors of hemozoin formation, the 1H-benzimidazole analogues also showed inhibitory effects on microtubules. In vivo efficacy studies in Plasmodium berghei-infected mice revealed that the frontrunner compound 41 exhibited high efficacy (98% reduction in parasitemia) when dosed orally at 4 × 50 mg/kg. Generally, the compounds were noncytotoxic to mammalian cells.

Malaria in livestock (cattle, buffalo, sheep, and goat) was detected and diagnosed in Mosul, Iraq. A total of forty infected animals of local breeds, from different ages and both sexes, showed Plasmodium parasites in their blood smears. Ten animals of each species were selected. All animals were clinically examined. The identification of Plasmodium parasites was done by microscopy and PCR assay. Fever, pale mucous membranes, lymphadenitis, jaundice, and recumbences are more common clinical signs were associated with malaria infection in livestock. Trophozoites, schizonts, and gametocytes appeared morphologically resemble those of Plasmodium spp. of humans. Sporozoites, Oocyte, and Ookinets were observed in stained blood smears of some cows, sheep, and goats. A high degree of parasitemia and erythrocytes infected with two or more trophozoites were seen. Trophozoites were seen more visible in the peripheral blood smears stained with the direct Acridine Orange and wet mount preparations in comparison to the traditional Giemsa, and Leishman stains method. The presence of hemozoin crystals was diagnostic of malaria parasites. Plasmodium DNA was detected from all samples. We need further studies to genotype Plasmodium spp. in livestock and to understand the prevalence of malaria parasites in animals of Iraq.

The continued emergence of resistance to front-line antimalarial treatments is of great concern. Therefore, new compounds that potentially have a novel target in various developmental stages of Plasmodium parasites are needed to treat patients and halt the spread of malaria. Here, several benzimidazole derivatives were screened for activity against the symptom-causing intraerythrocytic asexual blood stages and the transmissible gametocyte stages of P. falciparum. Submicromolar activity was obtained for 54 compounds against asexual blood stage parasites with 6 potent at IC50 < 100 nM while not displaying any marked toxicity against mammalian cells. Nanomolar potency was also observed against gametocytes with two compounds active against early stage gametocytes and two compounds active against late-stage gametocytes. The transmission-blocking potential of the latter was confirmed as they could prevent male gamete exflagellation and the lead compound reduced transmission by 72% in an in vivo mosquito feeding model. These compounds therefore have activity against multiple stages of Plasmodium parasites with potential for differential targets.

Solomon Yeshanew,1 Alemayehu Dagne,2 Behailu Taye2 1Department of Biology, Debre Markos University, Debre Markos, Ethiopia; 2Department of Biology, Mettu University, Mettu, EthiopiaCorrespondence: Solomon Yeshanew Tel +251-913-936-754Email solarm12@yahoo.comBackground: Chloroquine (CQ) is the first line treatment for vivax malaria in Ethiopia. However, the therapeutic efficacy of the drug is now declining. Several reports from different areas of the country showed CQ-vivax treatment failure increasing. This study therefore aimed to provide additional data on the therapeutic efficacy of CQ against Plasmodium vivax malaria from two districts of Southwestern Ethiopia.Methods: An observational prospective study among P. vivax malaria infected individuals was conducted in two districts of Southwest Ethiopia for a period of 28 follow-up days. Study participants were treated with 25 mg/kg of standard CQ for 3 consecutive days according to the procedure. Microscopic blood film examinations and other clinical assessments were measured within the follow-up period on a weekly basis.Results: A total of 115 patients were enrolled in the study. Sixty-five were from Darimu and 50 were from Bure districts. The majority (67%) of study participants were male and 86.1% (99/115) were below 35 years old. The study revealed that CQ treatment was able to clear vivax malaria parasites and febrile within a week. During the follow-up study period, recurrence of vivax parasitemia was not recorded. However, there was a marked heterogeneity with respect to fever clearance time, parasitemia load, and carriage of parasite gametocyte within 72 hours of post-treatment between the two study areas.Conclusion: The present study revealed that CQ has good clinical and parasitological response to vivax malaria in the study areas. Thus, it can be continued as the first line P. vivax malaria treatment. However, further monitoring and evaluation of the drug should be considered.Keywords: Chloroquine, In vivo, P. vivax, Southwest Ethiopia, Treatment failure

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.

A novel diazaspiro[3.4]octane series was identified from a Plasmodium falciparum whole-cell high-throughput screening campaign. Hits displayed activity against multiple stages of the parasite lifecycle, which together with a novel sp3-rich scaffold provided an attractive starting point for a hit-to-lead medicinal chemistry optimization and biological profiling program. Structure-activity-relationship studies led to the identification of compounds that showed low nanomolar asexual blood-stage activity (

Introduction: The development of effective vaccines remains a major health priority to combat the global burden of malaria, a life-threatening disease caused by Plasmodium parasites. Transmission-blocking vaccines (TBVs) elicit antibodies that neutralize the sexual stages of the parasite in blood meals ingested by the Anopheles mosquito, interrupting parasite development in the vector host and preventing disease spread to other individuals.Areas covered: The P. falciparum gametocyte surface antigens Pfs230, Pfs48/45, and Pfs47, the parasite ookinete surface protein Pfs25, and the male gametocyte specific protein PfHAP2 are leading TBV candidates, some of which are in clinical development. The recent expansion of methodology to study monoclonal antibodies isolated directly from humans and animal models, coupled with effective measures for parasite neutralization, has provided unprecedented insight into TBV efficacy and development.Expert opinion: Available structural and functional data on antigen-monoclonal antibody (Ag-mAb) complexes, as well as epitope classification studies, have identified neutralizing epitopes that may aid vaccine development and improve protection. Here, we review the clinical prospects of TBV candidates, progress in the development of novel vaccine strategies for TBVs, and the impact of structural vaccinology in TBV design.

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.

Background For malaria elimination efforts, it is important to better understand parasite transmission to mosquitoes and develop models for early-clinical evaluation of transmission-blocking interventions. Methods In a randomized open-label trial, 24 participants were infected by bites from Plasmodium falciparum 3D7-infected mosquitoes (mosquito bite [MB]; n = 12) or by induced blood-stage malaria (IBSM) with the same parasite line (n = 12). After subcurative piperaquine treatment, asexual parasite and gametocytes kinetics were assessed, and mosquito feeding experiments were performed. Results Study procedures were well tolerated. The median peak gametocyte density was 1304/mL (interquartile range, 308–1607/mL) after IBSM, compared with 14/mL (10–64/mL) after MB inoculation (P Conclusions We observed a striking effect of inoculation method on gametocyte production, suggesting higher gametocyte commitment after IBSM. Our direct comparison of MB and IBSM establishes the controlled human malaria infection transmission model, using intravenous administration of P. falciparum–infected erythrocytes as a model for early-clinical evaluation of interventions that aim to interrupt malaria transmission. Clinical Trial Registration NCT03454048

Plasmodium, the malaria parasite, undergoes a complex life cycle alternating between a vertebrate host and a mosquito vector of the genus Anopheles. In red blood cells of the vertebrate host, Plasmodium multiplies asexually or differentiates into gamete precursors, the male and female gametocytes, responsible for parasite transmission. Sexual stage maturation occurs in the midgut of the mosquito vector, where male and female gametes egress from the host erythrocytes to fuse and form a zygote. Gamete egress entails the successive rupture of two membranes surrounding the parasite, the parasitophorous vacuole membrane and the erythrocyte plasma membrane. In this study, we used the rodent model parasite Plasmodium berghei to design a label-free quantitative proteomic approach aimed at identifying gender-related proteins differentially released/secreted by purified mature gametocytes when activated to form gametes. We compared the abundance of molecules secreted by wild type gametocytes of both genders with that of a transgenic line defective in male gamete maturation and egress. This enabled us to provide a comprehensive data set of egress-related molecules and their gender specificity. Using specific antibodies, we validated eleven candidate molecules, predicted as either gender-specific or common to both male and female gametocytes. All of them localize to punctuate, vesicle-like structures that relocate to cell periphery upon activation, but only three of them localize to the gametocyte-specific secretory vesicles named osmiophilic bodies. Our results confirm that the egress process involves a tightly coordinated secretory apparatus that includes different types of vesicles and may put the basis for functional studies aimed at designing novel transmission-blocking molecules.

For malaria parasites balancing sexual commitment, the frequency with which asexual bloodstream forms differentiate into non-replicative male and female gametocytes is critical because asexual replication is required to maintain a persistent infection of the human host while the non-replicative gametocytes are essential for infection of the mosquito vector and transmission. Here we describe recent advances in understanding of the regulatory mechanisms controlling this key developmental decision. These include new insights into the mechanistic roles of the transcriptional master switch AP2-G and the epigenetic modulator GDV1, as well as the identification of defined metabolic signals that modulate their activity. Many of these metabolites are linked to parasite phospholipid biogenesis and we propose a model linking this pathway to the epigenetic regulation underlying sexual commitment in P. falciparum.

Malaria is spread by the transmission of sexual stage parasites, called gametocytes. However, with Plasmodium falciparum, gametocytes can only be detected in peripheral blood when they are mature and transmissible to a mosquito, which complicates control efforts. Here, we identify the set of genes overexpressed in patient blood samples with high levels of gametocyte-committed ring stage parasites. Expression of all 18 genes is regulated by transcription factor AP2-G, which is required for gametocytogenesis. We select three genes, not expressed in mature gametocytes, to develop as biomarkers. All three biomarkers we validate in vitro using 6 different parasite lines and develop an algorithm that predicts gametocyte production in ex vivo samples and volunteer infection studies. The biomarkers are also sensitive enough to monitor gametocyte production in asymptomatic P. falciparum carriers allowing early detection and treatment of infectious reservoirs, as well as the in vivo analysis of factors that modulate sexual conversion., Malaria gametocytes are sexual-stage parasites transmitted from mammalian host’s blood back to their insect vector. Here, Prajapati et al. identify gametocyte-committed ring-stage biomarkers allowing to forecast malaria transmission potential.

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.

Background Interventions that effectively target Plasmodium vivax are critical for the future control and elimination of malaria. We conducted a P. vivax volunteer infection study to characterize the antimalarial activity of artefenomel, a new drug candidate. Methods Eight healthy, malaria-naive participants were intravenously inoculated with blood-stage P. vivax and subsequently received a single oral 200-mg dose of artefenomel. Blood samples were collected to monitor the development and clearance of parasitemia, and plasma artefenomel concentration. Mosquito feeding assays were conducted before artefenomel dosing to investigate parasite transmissibility. Results Initial parasite clearance occurred in all participants after artefenomel administration (log10 parasite reduction ratio over 48 hours, 1.67; parasite clearance half-life, 8.67 hours). Recrudescence occurred in 7 participants 11–14 days after dosing. A minimum inhibitory concentration of 0.62 ng/mL and minimum parasiticidal concentration that achieves 90% of maximum effect of 0.83 ng/mL were estimated, and a single 300-mg dose was predicted to clear 109 parasites per milliliter with 95% certainty. Gametocytemia developed in all participants and was cleared 4–8 days after dosing. At peak gametocytemia, 75% of participants were infectious to mosquitoes. Conclusions The in vivo antimalarial activity of artefenomel supports its further clinical development as a treatment for P. vivax malaria. Clinical Trials Registration NCT02573857.

Malaria remains one of the most deadly infectious diseases, causing hundreds of thousands of deaths each year, primarily in young children and pregnant mothers. Here, we report the discovery and derivatization of a series of pyrazolo[3,4-b]pyridines targeting Plasmodium falciparum, the deadliest species of the malaria parasite. Hit compounds in this series display sub-micromolar in vitro activity against the intraerythrocytic stage of the parasite as well as little to no toxicity against the human fibroblast BJ and liver HepG2 cell lines. In addition, our hit compounds show good activity against the liver stage of the parasite but little activity against the gametocyte stage. Parasitological profiles, including rate of killing, docking, and molecular dynamics studies, suggest that our compounds may target the Qo binding site of cytochrome bc1.

Background Since the World Health Organization recommended single low-dose (0.25 mg/kg) primaquine (PQ) in combination with artemisinin-based combination therapies (ACTs) in areas of low transmission or artemisinin-resistant Plasmodium falciparum, several single-site studies have been conducted to assess efficacy. Methods An individual patient meta-analysis to assess gametocytocidal and transmission-blocking efficacy of PQ in combination with different ACTs was conducted. Random effects logistic regression was used to quantify PQ effect on (1) gametocyte carriage in the first 2 weeks post treatment; and (2) the probability of infecting at least 1 mosquito or of a mosquito becoming infected. Results In 2574 participants from 14 studies, PQ reduced PCR-determined gametocyte carriage on days 7 and 14, most apparently in patients presenting with gametocytemia on day 0 (odds ratio [OR], 0.22; 95% confidence interval [CI], .17–.28 and OR, 0.12; 95% CI, .08–.16, respectively). Rate of decline in gametocyte carriage was faster when PQ was combined with artemether-lumefantrine (AL) compared to dihydroartemisinin-piperaquine (DP) (P = .010 for day 7). Addition of 0.25 mg/kg PQ was associated with near complete prevention of transmission to mosquitoes. Conclusions Transmission blocking is achieved with 0.25 mg/kg PQ. Gametocyte persistence and infectivity are lower when PQ is combined with AL compared to DP.

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.

A within-human-host malaria parasite model, integrating key variables that influence parasite evolution-progression-advancement, under innate and adaptive immune responses, is analyzed. The implicit role of immunity on the steady state parasite loads and parasitemia reproduction number ([Formula: see text]), a threshold parameter measuring the parasite’s annexing ability of healthy red blood cells (HRBCs), eventually rendering a human infectious to mosquitoes, is investigated. The impact of the type of recruitment function used to model HRBC growth is also investigated. The model steady states and [Formula: see text], both obtained as functions of immune system variables, are analyzed at snapshots of immune sizes. Model results indicate that the more the immune cells, innate and adaptive, the more efficient they are at inhibiting parasite development and progression; consequently, the less severe the malaria disease in a patient. Our analysis also illustrates the existence of a Hopf bifurcation leading to a limit cycle, observable only for the nonlinear recruitment functions, at reasonably large [Formula: see text].

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.

Although the knowledge of morphometrics and hematology of reptiles in health has grown substantially in recent years, there are still knowledge gaps in many species and from different geographical regions. The objectives of this study were to document morphometrical measurements and hematological data of clinically healthy free-ranging adult Mesopotamian spiny-tailed lizards (Saara loricata), the Iraqi Mastigure or Iraqi spiny-tailed lizard, from Khuzestan Province, Iran, and to investigate sex differences in evaluated parameters. Although we did not observe any gender differences in morphometrical measurements, the visually larger genital pores of males allowed for differentiation of study animals into females (n = 6) and males (n = 5). We obtained femoral vein blood samples for hematological analysis, including detailed red blood cell (RBC) measurements, RBC count, packed cell volume (PCV), white blood cell (WBC) counts, and blood film review. Gender differences were identified in some RBC measurements, notably length and width of erythrocytes and nucleus width, as well as RBC and WBC counts. Gametocytes of hemogregarines varied from absent to frequent. The information presented herein will be useful for interpretative considerations of health assessment data for this species from Iran in comparison to data from the same and closely related species from other geographical regions.

A possible malaria control approach involves the dissemination in mosquitoes of inherited symbiotic microbes to block Plasmodium transmission. However, in the Anopheles gambiae complex, the primary African vectors of malaria, there are limited reports of inherited symbionts that impair transmission. We show that a vertically transmitted microsporidian symbiont (Microsporidia MB) in the An. gambiae complex can impair Plasmodium transmission. Microsporidia MB is present at moderate prevalence in geographically dispersed populations of An. arabiensis in Kenya, localized to the mosquito midgut and ovaries, and is not associated with significant reductions in adult host fecundity or survival. Field-collected Microsporidia MB infected An. arabiensis tested negative for P. falciparum gametocytes and, on experimental infection with P. falciparum, sporozoites aren’t detected in Microsporidia MB infected mosquitoes. As a microbe that impairs Plasmodium transmission that is non-virulent and vertically transmitted, Microsporidia MB could be investigated as a strategy to limit malaria transmission., Mircobial symbionts of mosquitoes can affect transmission of human pathogens. Here, Herren et al. identify a microsporidian symbiont in Anopheles gambiae that impairs transmission without affecting mosquito fecundity or survival.

BACKGROUNDInterventions that interrupt Plasmodium vivax transmission or eliminate dormant P. vivax liver-stage parasites will be essential for malaria elimination. Development of these interventions has been hindered by the lack of P. vivax in vitro culture and could be accelerated by a safe and reproducible clinical model in malaria-naive individuals.METHODSHealthy, malaria-naive adults were enrolled in 2 studies to assess the safety, infectivity, and transmissibility of a new P. vivax isolate. Participants (Study 1, n = 2; Study 2, n = 24) were inoculated with P. vivax-infected red blood cells to initiate infection, and were treated with artemether-lumefantrine (Study 1) or chloroquine (Study 2). Primary endpoints were safety and infectivity of the new isolate. In Study 2, transmission to mosquitoes was also evaluated using mosquito feeding assays, and sporozoite viability was assessed using in vitro cultured hepatocytes.RESULTSParasitemia and gametocytemia developed in all participants and was cleared by antimalarial treatment. Adverse events were mostly mild or moderate and none were serious. Sixty-nine percent of participants (11/16) were infectious to Anopheles mosquitoes at peak gametocytemia. Mosquito infection rates reached 97% following membrane feeding with gametocyte-enriched blood, and sporozoites developed into liver-stage schizonts in culture.CONCLUSIONWe have demonstrated the safe, reproducible, and efficient transmission of P. vivax gametocytes from humans to mosquitoes, and have established an experimental model that will accelerate the development of interventions targeting multiple stages of the P. vivax life cycle.TRIAL REGISTRATIONACTRN12614000930684 and ACTRN12616000174482.FUNDING(Australian) National Health and Medical Research Council Program Grant 1132975 (Study 1). Bill and Melinda Gates Foundation (OPP1111147) (Study 2).

Purpose Continuous efforts into the discovery and development of new antimalarials are required to face the emerging resistance of the parasite to available treatments. Thus, new effective drugs, ideally able to inhibit the Plasmodium life-cycle stages that cause the disease as well as those responsible for its transmission, are needed. Eight compounds from the Medicines for Malaria Venture (MMV) Malaria Box, potentially interfering with the parasite polyamine biosynthesis were selected and assessed in vitro for activity against malaria transmissible stages, namely mature gametocytes and early sporogonic stages. Methods Compound activity against asexual blood stages of chloroquine-sensitive 3D7 and chloroquine-resistant W2 strains of Plasmodium falciparum was tested measuring the parasite lactate dehydrogenase activity. The gametocytocidal effect was determined against the P. falciparum 3D7elo1-pfs16-CBG99 strain with a luminescent method. The murine P. berghei CTRP.GFP strain was employed to assess compounds activities against early sporogonic stage development in an in vitro assay simulating mosquito midgut conditions. Results Among the eight tested molecules, MMV000642, MMV000662 and MMV006429, containing a 1,2,3,4-tetrahydroisoquinoline-4-carboxamide chemical skeleton substituted at N-2, C-3 and C-4, displayed multi-stage activity. Activity against asexual blood stages of both strains was confirmed with values of IC50 (50% inhibitory concentration) in the range of 0.07-0.13 µM. They were also active against mature stage V gametocytes with IC50 values below 5 µM (range: 3.43-4.42 µM). These molecules exhibited moderate effects on early sporogonic stage development, displaying IC50 values between 20 and 40 µM. Conclusion Given the multi-stage, transmission-blocking profiles of MMV000642, MMV000662, MMV006429, and their chemical characteristics, these compounds can be considered worthy for further optimisation toward a TCP5 or TCP6 target product profile proposed by MMV for transmission-blocking antimalarials.

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.

This study aims to evaluate the therapeutic efficacy and tolerance of two ACTs widely used for the treatment of uncomplicated malaria due to Plasmodium falciparum in Niger. The study was conducted from September to November 2017, at the Integrated Health Centers of Dogondoutchi and Birni N'Gaoure, in patients aged from 6 months to 15 years, with uncomplicated malaria due to Plasmodium falciparum. They were treated with either Artemether-Lumefantrine (AL) or Artesunate-Amodiaquine (ASAQ). The primary endpoint was the appropriate clinical and parasitological response (RCPA) to D28, after PCR correction. The secondary criteria were the clearing time of fever, parasites, and gametocytes and then the occurrence of adverse events. A total of 459 patients were examined, of whom 312 patients met the inclusion criteria for therapeutic efficacy evaluation. We have followed 299 patients up to J28 including 146 in the AL arm and 153 in the ASAQ arm. After PCR correction at J28, RCPA were 95.8% and 96% (P = 0.7185) for arms AL and ASAQ, respectively, compared to 93.1% and 94.1% respectively before PCR correction (P = 0.7892). The number of patients on AL and ASAQ treatment who developed an adverse reaction were 6 (7.6%) and 23 (28%) respectively. AL and ASAQ associations are effective and well tolerated. No serious adverse event was noted. However, their monitoring must continue to detect possible resistance.

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: More than 90% of the global 400 000 annual malaria deaths occur in Africa. The current SARS-CoV-2 pandemic has resulted in more than 830 000 deaths in its first 10 months. Case presentation: This case describes a patient who had travelled from Mozambique to Cape Town, presented with a mild febrile illness, and was diagnosed with both COVID-19 and uncomplicated Plasmodium falciparum malaria infection. She responded well to malaria treatment and had an uneventful COVID-19 admission. Her blood smear showed a low malaria parasitaemia and a relatively high gametocyte load. Conclusion: We postulate that her clinical course and abnormal smear could well be due to reciprocal disease-modifying effects of the infections. The presenting symptoms of COVID-19 may mimic endemic infectious diseases including malaria, tuberculosis, pneumocystis pneumonia and influenza thus there is a need for clinical vigilance to identify and treat such co-infections. The full article is available at https://doi.org/10.36303/JMLSTSA.2020.2.2.57

Malaria, a global threat to the human population, remains a challenge partly due to the fast-growing drug-resistant strains of Plasmodium species. New therapeutics acting against the pathogenic asexual and sexual stages, including liver-stage malarial infection, have now attained more attention in achieving malaria eradication efforts. In this paper, two previously identified potent antiplasmodial hydroxyethylamine (HEA) compounds were investigated for their activity against the malaria parasite's multiple life stages. The compounds exhibited notable activity against the artemisinin-resistant strain of P. falciparum blood-stage culture with 50% inhibitory concentrations (IC50) in the low micromolar range. The compounds' cytotoxicity on HEK293, HepG2 and Huh-7 cells exhibited selective killing activity with IC50 values > 170 μM. The in vivo efficacy was studied in mice infected with P. berghei NK65, which showed a significant reduction in the blood parasite load. Notably, the compounds were active against liver-stage infection, mainly compound 1 with an IC50 value of 1.89 μM. Mice infected with P. berghei sporozoites treated with compound 1 at 50 mg kg−1 dose had markedly reduced liver stage infection. Moreover, both compounds prevented ookinete maturation and affected the developmental progression of gametocytes. Further, systematic in silico studies suggested both the compounds have a high affinity towards plasmepsin II with favorable pharmacological properties. Overall, the findings demonstrated that HEA and piperidine possessing compounds have immense potential in treating malarial infection by acting as multistage inhibitors.

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 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: In India, the epidemiology of malaria is complex because of geo-ecological diversity, multi-ethnicity, and wide distribution of nine anopheline vectors transmitting two commonest plasmodia species. Considering the fact, it is vital that every centre has its own demographic and pathological data about the profile of malaria patients in its catchment area. Methods: The present retrospective study was carried in the Department of Pathology, Gujarat Adani Institute of Medical Sciences (GAIMS), Bhuj for a period of one year. A total of 102 cases were included in the study. The blood samples were collected in Ethylenediamine tetra-acetic acid (EDTA) vacutainers and smears were prepared. Leishman staining and field staining on thick smears was done according to the published protocols. The diagnosis of malaria parasite was confirmed by a positive peripheral smear examination. Rings, schizonts and gametocytes of the malaria parasites (plasmodium vivax and plasmodium falciparum) were viewed for diagnosis. Thrombocytopenia was considered when the platelet count was less than 150 x 109/L and leucopenia when the total leucocyte count was less than 4,000 cells /cu mm. Result: In our study, 102 malaria positive cases were investigated for platelet count and total leucocyte count. Out of the 102 cases, male population was affected more than female. Out of the various malaria species, plasmodium vivax was the most prevalent (82.35%). The most common age group affected by various species varied, with age group of 1-10 years being most common for plasmodium falciparum cases, 21 to 30 years being most common for plasmodium vivax and 11 to 20 years being most common for mixed infection. All the cases of plasmodium falciparum were found to be with normal leucocyte count whereas in plasmodium vivax 82.14% cases were with normal leucocyte count and only 17.85% cases with leucopenia. Mixed infection had almost similar scenario with 71.42% cases with normal leucocyte count and 28.57% with leucopenia. In our study more number of cases of plasmodium falciparum was associated with thrombocytopenia (25%) as compared to plasmodium vivax (11.9%) and none in mixed infection. Conclusion: The high prevalence of malaria suggests the importance of timely diagnosis . The early identification of thrombocytopenia and leucopenia aids in timely management. This helps to decrease complicated malaria cases and its related mortality.

Gametocytes of the malaria parasite Plasmodium are taken up by the mosquito vector with an infectious blood meal, representing a critical stage for parasite transmission. Calcium-independent protein kinases (CDPKs) play key roles in calcium-mediated signaling across the complex life cycle of the parasite. We sought to understand their role in human parasite transmission from the host to the mosquito vector and thus investigated the role of the human-infective parasite Plasmodium falciparum CDPK4 in the parasite life cycle. P. falciparum cdpk4− parasites created by targeted gene deletion showed no effect in blood stage development or gametocyte development. However, cdpk4− parasites showed a severe defect in male gametogenesis and the emergence of flagellated male gametes. To understand the molecular underpinnings of this defect, we performed mass spectrometry-based phosphoproteomic analyses of wild-type and Plasmodium falciparum cdpk4− late gametocyte stages to identify key CDPK4-mediated phosphorylation events that may be important for the regulation of male gametogenesis. We further employed in vitro assays to identify these putative substrates of Plasmodium falciparum CDPK4. This indicated that CDPK4 regulates male gametogenesis by directly or indirectly controlling key essential events, such as DNA replication, mRNA translation, and cell motility. Taken together, our work demonstrates that PfCDPK4 is a central kinase that regulates exflagellation and thereby is critical for parasite transmission to the mosquito vector. IMPORTANCE Transmission of the malaria parasite to the mosquito vector is critical for the completion of the sexual stage of the parasite life cycle and is dependent on the release of male gametes from the gametocyte body inside the mosquito midgut. In the present study, we demonstrate that PfCDPK4 is critical for male gametogenesis and is involved in phosphorylation of proteins essential for male gamete emergence. Targeting PfCDPK4 and its substrates may provide insights into achieving effective malaria transmission-blocking strategies.

Blocking malaria transmission is critical to malaria control programs but remains a major challenge especially in endemic regions with high levels of asymptomatic infections. New strategies targeting the transmissible sexual stages of the parasite, called gametocytes, are needed. This review focuses on P. falciparum gametocytogenesis in vivo and in vitro. Highlighting advances made elucidating genes required for gametocyte production and identifying key questions that remain unanswered such as the factors and regulatory mechanisms that contribute to gametocyte induction, and the mechanism of sequestration. Tools available to begin to address these issues are also described to facilitate advances in our understanding of this important stage of the life cycle.

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.

Despite being a weak point in their life cycle, transmission of Plasmodium parasites from humans to mosquitoes is an understudied field of research. Direct membrane feeding assays (DMFA) are an important tool, allowing detailed mechanistic malaria transmission studies from humans to mosquitoes. Especially for Plasmodium vivax, which cannot be cultured long-term under laboratory conditions, implementation of DMFAs requires proximity to P. vivax endemic areas. In the present study, we investigated the infectivity of symptomatic Plasmodium infections to Anopheles farauti colony mosquitoes in Papua New Guinea (PNG), a country with one of the highest rates of Plasmodium vivax in the world. A total of 182 DMFAs were performed with venous blood collected from symptomatic malaria patients positive by rapid diagnostic test (RDT). DMFAs resulted in mosquito infection in 20.9% (38/182) of cases. The parasite species in the blood feeds were determined retrospectively by expert light microscopy and quantitative real-time qPCR. Based on light microscopy, 9.2% of P. falciparum and 42% of P. vivax human infections resulted in mosquito infections. Infections containing gametocytes detected by microscopy led to mosquito infections in 58.8% of P. vivax and 8.7% of P. falciparum infections. Based on qPCR, 10% of P. falciparum and 43.6% of P. vivax lead to a successful mosquito infection. Venous blood samples from symptomatic P. vivax patients were more infectious to An. farauti mosquitoes in DMFAs compared to P. falciparum infected patients. The capacity to perform DMFAs in a high-burden P. vivax setting creates a unique opportunity to address critical gaps in our understanding of P. vivax human-tomosquito transmission.

Malaria parasites rely on specialized stages, called gametocytes, to ensure human-to-human transmission. The formation of these sexual precursor cells is initiated by commitment of blood stage parasites to the sexual differentiation pathway. Plasmodium falciparum, the most virulent of six parasite species infecting humans, employs nutrient sensing to control the rate at which sexual commitment is initiated, and the presence of stress-inducing factors, including antimalarial drugs, has been linked to increased gametocyte production in vitro and in vivo. These observations suggest that therapeutic interventions may promote gametocytogenesis and malaria transmission. Here, we engineered a P. falciparum reporter line to quantify sexual commitment rates after exposure to antimalarials and other pharmaceuticals commonly prescribed in malaria-endemic regions. Our data reveal that some of the tested drugs indeed have the capacity to elevate sexual commitment rates in vitro. Importantly, however, these effects are only observed at drug concentrations that inhibit parasite survival and only rarely result in a net increase of gametocyte production. Using a drug-resistant parasite reporter line, we further show that the gametocytogenesis-promoting effect of drugs is linked to general stress responses rather than to compound-specific activities. Altogether, we provide conclusive evidence for the absence of mechanistic links between the regulation of sexual commitment and the activity of commonly used pharmaceuticals in vitro. Our data hence contradict scenarios in which therapeutic interventions would promote the spread of drug-resistant parasites or malaria transmission in general.

BackgroundIn areas where Plasmodium falciparum malaria is highly seasonal, a dry season reservoir of blood-stage infection is essential for initiating transmission during the following wet season, bridging transmission seasons several months apart. Understanding infections during the dry season could thus inform approaches for malaria control.MethodsIn The Gambia, a cohort of 42 individuals with qPCR positive P. falciparum infections at the end of the transmission season (December) were followed monthly until the end of the dry season (May) to evaluate the duration of detectable infections. The influence of human host (age, sex, haemoglobin concentration and genotype, and P. falciparum-specific antibodies), and parasitological (parasite density, gametocyte density and genotypic multiplicity of infection) factors was investigated.ResultsA large proportion of individuals infected at the end of the wet season had detectable infections until the end of the dry season (40.0%; 16/40), with the majority of these infections also harbouring gametocytes (81.3%; 13/16). 22 infections were classified as persistent (detectable for at least 3 months), 17 were classified as short-lived (undetectable within 2 months), and 3 were treated (due to symptoms). At the start of the dry season, the majority of persistent infections (82%; 18/22) had parasite densities >10 p/µL compared to only 5.9% (1/17) of short-lived infections. Persistent infections (59%; 13/22) were also more likely to be multi-clonal than short-lived infections (5.9%; 1/17), they were most common in 5 to 15 year old children (63%; 12/19), and were associated with individuals having higher levels of P. falciparum-specific antibodies (p = 0.058).ConclusionsAsymptomatic persistent dry season infections in The Gambia were multiclonal with higher parasite densities at the beginning of the dry season, mostly occurring in school age children and adults with higher P. falciparum-specific antibodies. Screening and treating asymptomatic, malaria-infected individuals during the dry season may reduce the human reservoir of malaria responsible initiating transmission in the wet-season.