Stage Plasmodium Research Articles

Plasmodium Falciparum Rifins: Role in malaria pathogenesis

Malaria kills an estimated 600,000 people each year, especially children under five years who reside in sub-Saharan Africa. Malaria fatalities are associated with severe forms such as cerebral malaria, acute respiratory failure, severe anaemia, renal failure, hypoglycaemia, and pulmonary oedema. Although the underlying pathogenic mechanisms in immune responses and parasite immune evasion, cytoadherence of parasitized red blood cells, and rosetting are enumerated, the mechanisms are not fully understood. P. falciparum parasite-derived surface protein, repetitive interspersed family (RIFIN) genes are involved in rosetting, blocking microcirculation, and playing a role in malaria pathogenesis; it is unclear which RIFIN family genes are involved in the various pathogenic mechanisms in malaria. RIFINs are the extensive malaria family genes expressed throughout the malaria parasite stages, indicating their diverse roles. Malaria pathogenesis occurs in erythrocyte-stage infection, and the expression of RIFINs at this phase could play a diverse role in the various pathogenic mechanisms. They are involved in major phenomena such as cytoadherence, merozoite evasion, and immune evasion. RIFINs aid in the immune evasion of P. falciparum through various molecular interactions by binding to the inhibitory receptors LAIR1, LILRB1, and LILRB2. RIFINs in severe forms of malaria (such as cerebral malaria and severe anaemia) require a considerable understanding to target and control malaria severity and mortality. RIFINs are implicated in severe malaria and are discussed together with other variant surface antigens such as STEVORS or PfEMP1 in the specific pathophysiology of malaria. This review details the role of RIFINs in the various malaria pathophysiological mechanisms underlying severe malaria and mortality

Linking avian malaria parasitemia estimates from quantitative PCR and microscopy reveals new infection patterns in Hawai'i

Plasmodium parasites infect thousands of species and provide an exceptional system for studying host-pathogen dynamics, especially for multi-host pathogens. However, understanding these interactions requires an accurate assay of infection. Assessing Plasmodium infections using microscopy on blood smears often misses infections with low parasitemias (the fractions of cells infected), and biases in malaria prevalence estimates will differ among hosts that differ in mean parasitemias. We examined Plasmodium relictum infection and parasitemia using both microscopy of blood smears and quantitative polymerase chain reaction (qPCR) on 299 samples from multiple bird species in Hawai'i and fit models to predict parasitemias from qPCR cycle threshold (Ct) values. We used these models to quantify the extent to which microscopy underestimated infection prevalence and to more accurately estimate infection patterns for each species for a large historical study done by microscopy. We found that most qPCR-positive wild-caught birds in Hawaii had low parasitemias (Ct scores ≥35), which were rarely detected by microscopy. The fraction of infections missed by microscopy differed substantially among eight species due to differences in species’ parasitemia levels. Infection prevalence was likely 4–5-fold higher than previous microscopy estimates for three introduced species, including Zosterops japonicus, Hawaii’s most abundant forest bird, which had low average parasitemias. In contrast, prevalence was likely only 1.5–2.3-fold higher than previous estimates for Himatione sanguinea and Chlorodrepanis virens, two native species with high average parasitemias. Our results indicate that relative patterns of infection among species differ substantially from those observed in previous microscopy studies, and that differences depend on variation in parasitemias among species. Although microscopy of blood smears is useful for estimating the frequency of different Plasmodium stages and host attributes, more sensitive quantitative methods, including qPCR, are needed to accurately estimate and compare infection prevalence among host species.

Novel thienopyrimidones targeting hepatic and erythrocytic stages of Plasmodium parasites with increased microsomal stability

Based on the structure of a previously identified hit, Gamhepathiopine 1, which showed promising antiplasmodial activity, but poor microsomal stability, several strategies were investigated to improve the metabolic stability of the compounds. This included the introduction of fluorine or deuterium atoms, as well as carbocyclic groups. Among the new compounds, the 2-aminocyclobutyl derivative 5g demonstrated enhanced microsomal stability compared to compound 1, while retaining antiplasmodial activity against erythrocytic and hepatic stages of Plasmodium, without significant cytotoxicity against primary hepatocytes.

Annickia affinis (Exell) Versteegh & Sosef methanol stem bark extract, potent fractions and isolated Berberine alkaloid target both blood and liver stages of malaria parasites

Ethnopharmacological relevanceHaving identified Annickia affinis as the most potent antiplasmodial plant constituent in a hepta-herbal Agbo-iba (HHA) formula commonly used to manage malaria in Benin city, Nigeria, we have in this study attempted to identify the specialized metabolites responsible for antiplasmodial activity of A. affinis through anti-blood stage malaria parasite activity guided isolation of potent molecules from its stem bark methanol extract. After that, phenotypic effects, including stage-specific kill kinetics, were investigated. Further, the crude extract, its potent fractions, and specialized metabolites were also tested against the liver-stage malaria parasite. Materials and methodsA. affinis was subjected to molecular PCR-based analysis to confirm its identity. Thereafter, extraction of its stem bark with methanol was carried out. Alkaloid enriched fractions from this stem bark extract were obtained using the acid-base-solvent extraction method. These alkaloid-enriched fractions were subjected to various chromatographic techniques that led to the isolation of two protoberberine alkaloids identified as berberine and palmatine based on NMR and mass spectrometry analysis. The efficacy of crude extract, fractions and purified alkaloids was tested against the malaria parasite's blood and liver stages, respectively. Results and DiscussionAnnickia affinis methanol extract, fractions, and the isolated protoberberine alkaloids showed excellent antiplasmodial activity with good selectivity against blood-stage malaria parasite. Thus, their IC50 against various strains of the parasite ranged from 0.95 to 18.65 μg/ml, while CC50 against Human embryonic kidney (HEK) and the human hepatoma (HUH-7) cell lines ranged between 10 and > 100 μg/ml. Interestingly, the crude extract and the alkaloid enriched fractions showed promising activity against the liver-stage malaria parasite. Between berberine and palmatine isolated from the potent fractions, only the former showed ∼100% and 90% inhibitions of liver stage parasite at 5 μg/ml and 1 μg/ml, respectively, while the latter showed no inhibition even at 20 μg/ml. ConclusionThis study reports that the ethnomedicinal use of HHA to manage malaria can be attributed to the presence of promising antiplasmodial protoberberine alkaloids together with synergistic effects via either enhancement of bioavailability or improved pharmacokinetics by other phytoconstituent(s) coming from other HHA constituent plants. The protoberberine alkaloids isolated have been identified as fast-acting antiplasmodial agents, with activity against all erythrocytic stages of the malaria parasite. Further, A. affinis methanol stembark extract and the protoberberine alkaloid berberine isolated from it also displayed excellent activity (>90% inhibition at 1 μg/ml) against the liver-stage malaria parasite. A. affinis and HHA can thus be useful as both liver-stage prophylactic and blood-stage curative agents.

Immunological characteristics of CD103+CD8+ Tc cells in the liver of C57BL/6 mouse infected with plasmodium NSM.

CD103 is an important marker of tissue-resident memory T cells (TRM) which play important roles in fighting against infection. However, the immunological characteristics of CD103+ T cells are not thoroughly elucidated in the liver of mouse infected with Plasmodium. Six- to eight-week-old C57BL/6 mice were infected with Plasmodium yoelii nigeriensis NSM. Mice were sacrificed on 12-16days after infection and the livers were picked out. Sections of the livers were stained, and serum aspartate aminotransferase (AST) and alanine transaminase (ALT) levels were measured. Moreover, lymphocytes in the liver were isolated, and the expression of CD103 was determined by using qPCR. The percentage of CD103 on different immune cell populations was dynamically observed by using flow cytometry (FCM). In addition, the phenotype and cytokine production characteristics of CD103+CD8+ Tc cell were analyzed by using flow cytometry, respectively. Erythrocyte stage plasmodium infection could result in severe hepatic damage, a widespread inflammatory response and the decrease of CD103 expression on hepatic immune cells. Only CD8+ Tc and γδT cells expressed higher levels of CD103 in the uninfected state.CD103 expression in CD8+ Tc cells significantly decreased after infection. Compared to that of CD103- CD8+ Tc cells, CD103+ CD8+ Tc cells from the infected mice expressed lower level of CD69, higher level of CD62L, and secreted more IL-4, IL-10, IL-17, and secreted less IFN-γ. CD103+CD8+ Tc cells might mediate the hepatic immune response by secreting IL-4, IL-10, and IL-17 except IFN-γ in the mice infected with the erythrocytic phase plasmodium, which could be involved in the pathogenesis of severe liver damage resulted from the erythrocytic phase plasmodium yoelii nigeriensis NSM infection.

Structural Analysis of Plasmodium falciparum Hexokinase Provides Novel Information about Catalysis Due to a Plasmodium-Specific Insertion.

The protozoan parasite Plasmodium falciparum is the causative pathogen of the most severe form of malaria, for which novel strategies for treatment are urgently required. The primary energy supply for intraerythrocytic stages of Plasmodium is the production of ATP via glycolysis. Due to the parasite's strong dependence on this pathway and the significant structural differences of its glycolytic enzymes compared to its human counterpart, glycolysis is considered a potential drug target. In this study, we provide the first three-dimensional protein structure of P. falciparum hexokinase (PfHK) containing novel information about the mechanisms of PfHK. We identified for the first time a Plasmodium-specific insertion that lines the active site. Moreover, we propose that this insertion plays a role in ATP binding. Residues of the insertion further seem to affect the tetrameric interface and therefore suggest a special way of communication among the different monomers. In addition, we confirmed that PfHK is targeted and affected by oxidative posttranslational modifications (oxPTMs). Both S-glutathionylation and S-nitrosation revealed an inhibitory effect on the enzymatic activity of PfHK.

Species composition, infection rate and detection of resistant alleles in Anopheles funestus (Diptera: Culicidae) from Lare, a malaria hotspot district of Ethiopia

BackgroundAnopheles funestus, which is considered as secondary vector of malaria in Ethiopia, is known to have several morphologically indistinguishable (sibling) species. Accurate identification of sibling species is crucial to understand their biology, behaviour and vector competence. In this study, molecular identification was conducted on the Ethiopian An. funestus populations. Moreover, insecticide resistance mechanism markers were detected, including ace N485I, kdr L1014F, L1014S, and CYP6P9a TaqMan qPCR was used to detect the infective stage of the parasite from field collected adult female An. funestus populations.MethodsAdult female mosquito collection was conducted from Lare, Gambella Regional State of Ethiopia between June 2018 to July 2020 using CDC light traps and HLC. Sub-samples of the morphologically identified An. funestus mosquitoes were molecularly identified using species-specific PCR, and the possible presence of insecticide resistance alleles was investigated using TaqMan qPCR (N485I-Ace-1), PCR-Sanger sequencing (L1014F-kdr), and PCR–RFLP (CYP6P9a resistance allele). Following head/thorax dissection, the TaqMan qPCR assay was used to investigate the presence of the infective stage Plasmodium parasite species.ResultsA total of 1086 adult female An. funestus mosquitoes were collected during the study period. All sub-samples (N = 20) that were morphologically identified as An. funestus sensu lato (s.l.) were identified as An. funestus sensu stricto (s.s.) using species- specific PCR assay. The PCR–RFLP assay that detects the CYP6P9a resistance allele that confers pyrethroid resistance in An. funestus was applied in N = 30 randomly selected An. funestus s.l. specimens. None of the specimens showed a digestion pattern consistent with the presence of the CYP6P9a resistance allele in contrast to what was observed in the positive control. Consequently, all samples were characterized as wild type. The qPCR TaqMan assay that detects the N485I acetylcholinesterase-1 mutation conferring resistance to organophosphates/carbamates in An. funestus was used in (N = 144) samples. All samples were characterized as wild type. The kdr L1014F and L1014S mutations in the VGSC gene that confer resistance to pyrethroids and DDT were analysed with direct Sanger sequencing after PCR and clean-up of the PCR products were also characterized as wild type. None of the samples (N = 169) were found positive for Plasmodium (P. falciparum/ovale/malariae/vivax) detection.ConclusionAll An. funestus s.l. samples from Lare were molecularly identified as An. funestus s.s. No CYP6P9, N485I acetylcholinesterase 1, kdr L1014F or L1014S mutations were detected in the An. funestus samples. None of the An. funestus samples were positive for Plasmodium. Although the current study did not detect any insecticide resistant mechanism, it provides a reference for future vector monitoring programmes. Regular monitoring of resistance mechanisms covering wider geographical areas of Ethiopia where this vector is distributed is important for improving the efficacy of vector control programs.

Plasmodium berghei oocysts possess fatty acid synthesis and scavenging routes

Malaria parasites carry out fatty acid synthesis (FAS) in their apicoplast organelle via a bacterially related (type II) enzymatic pathway. In the vertebrate host, exoerythrocytic Plasmodium stages rely on FAS, whereas intraerythrocytic stages depend on scavenging FA from their environment. In the mosquito, P. falciparum oocysts express and rely on FAS enzymes for sporozoite formation, but P. yoelii oocysts do not express, nor depend on, FAS enzymes and thus rely on FA scavenging to support sporogony. In P. berghei, FAS enzymes are similarly expendable for sporogony, indicating it conforms to the P. yoelii scenario. We show here that P. berghei, unexpectedly, expresses FAS enzymes throughout oocyst development. These findings indicate that P. berghei can employ FAS alongside FA scavenging to maximise sporogony and transmission, and is more similar to P. falciparum than previously assumed with respect to FA acquisition by the oocyst. The ability of oocysts to switch between FAS and scavenging could be an important factor in the non-competitive relationship of resource exploitation between Plasmodium parasites and their mosquito vectors, which shapes parasite virulence both in the insect and vertebrate.

Facile Access to Structurally Diverse Antimalarial Indoles Using a One-Pot A3 Coupling and Domino Cyclization Approach.

A multistep and diversity-oriented synthetic route aiming at the A3 coupling/domino cyclization of o-ethynyl anilines, aldehydes and s-amines is described. The preparation of the corresponding precursors included a series of transformations, such as haloperoxidation and Sonogashira cross-coupling reactions, amine protection, desilylation and amine reduction. Some products of the multicomponent reaction underwent further detosylation and Suzuki coupling. The resulting library of structurally diverse compounds was evaluated against blood and liver stage malaria parasites, which revealed a promising lead with sub-micromolar activity against intra-erythrocytic forms of Plasmodium falciparum. The results from this hit-to-lead optimization are hereby reported for the first time.

A malaria parasite phospholipase facilitates efficient asexual blood stage egress.

Malaria parasite release (egress) from host red blood cells involves parasite-mediated membrane poration and rupture, thought to involve membrane-lytic effector molecules such as perforin-like proteins and/or phospholipases. With the aim of identifying these effectors, we disrupted the expression of two Plasmodium falciparum perforin-like proteins simultaneously and showed that they have no essential roles during blood stage egress. Proteomic profiling of parasite proteins discharged into the parasitophorous vacuole (PV) just prior to egress detected the presence in the PV of a lecithin:cholesterol acyltransferase (LCAT; PF3D7_0629300). Conditional ablation of LCAT resulted in abnormal egress and a reduced replication rate. Lipidomic profiles of LCAT-null parasites showed drastic changes in several phosphatidylserine and acylphosphatidylglycerol species during egress. We thus show that, in addition to its previously demonstrated role in liver stage merozoite egress, LCAT is required to facilitate efficient egress in asexual blood stage malaria parasites.

Activity of Ivermectin and Its Metabolites against Asexual Blood Stage Plasmodium falciparum and Its Interactions with Antimalarial Drugs.

Ivermectin is an endectocide used widely to treat a variety of internal and external parasites. Field trials of ivermectin mass drug administration for malaria transmission control have demonstrated a reduction of Anopheles mosquito survival and human malaria incidence. Ivermectin will mostly be deployed together with artemisinin-based combination therapies (ACT), the first-line treatment of falciparum malaria. It has not been well established if ivermectin has activity against asexual stage Plasmodium falciparum or if it interacts with the parasiticidal activity of other antimalarial drugs. This study evaluated antimalarial activity of ivermectin and its metabolites in artemisinin-sensitive and artemisinin-resistant P. falciparum isolates and assessed in vitro drug-drug interaction with artemisinins and its partner drugs. The concentration of ivermectin causing half of the maximum inhibitory activity (IC50) on parasite survival was 0.81 μM with no significant difference between artemisinin-sensitive and artemisinin-resistant isolates (P = 0.574). The ivermectin metabolites were 2-fold to 4-fold less active than the ivermectin parent compound (P < 0.001). Potential pharmacodynamic drug-drug interactions of ivermectin with artemisinins, ACT-partner drugs, and atovaquone were studied in vitro using mixture assays providing isobolograms and derived fractional inhibitory concentrations. There were no synergistic or antagonistic pharmacodynamic interactions when combining ivermectin and antimalarial drugs. In conclusion, ivermectin does not have clinically relevant activity against the asexual blood stages of P. falciparum. It also does not affect the in vitro antimalarial activity of artemisinins or ACT-partner drugs against asexual blood stages of P. falciparum.

Isoliensinine from Cissampelos pariera rhizomes exhibits potential gametocytocidal and anti-malarial activities against Plasmodium falciparum clinical isolates

BackgroundThe unmet demand for effective malaria transmission-blocking agents targeting the transmissible stages of Plasmodium necessitates intensive discovery efforts. In this study, a bioactive bisbenzylisoquinoline (BBIQ), isoliensinine, from Cissampelos pariera (Menispermaceae) rhizomes was identified and characterized for its anti-malarial activity.MethodsMalaria SYBR Green I fluorescence assay was performed to evaluate the in vitro antimalarial activity against D6, Dd2, and F32-ART5 clones, and immediate ex vivo (IEV) susceptibility for 10 freshly collected P. falciparum isolates. To determine the speed- and stage-of-action of isoliensinine, an IC50 speed assay and morphological analyses were performed using synchronized Dd2 asexuals. Gametocytocidal activity against two culture-adapted gametocyte-producing clinical isolates was determined using microscopy readouts, with possible molecular targets and their binding affinities deduced in silico.ResultsIsoliensinine displayed a potent in vitro gametocytocidal activity at mean IC50gam values ranging between 0.41 and 0.69 µM for Plasmodium falciparum clinical isolates. The BBIQ compound also inhibited asexual replication at mean IC50Asexual of 2.17 µM, 2.22 µM, and 2.39 µM for D6, Dd2 and F32-ART5 respectively, targeting the late-trophozoite to schizont transition. Further characterization demonstrated a considerable immediate ex vivo potency against human clinical isolates at a geometric mean IC50IEV = 1.433 µM (95% CI 0.917–2.242). In silico analyses postulated a probable anti-malarial mechanism of action by high binding affinities for four mitotic division protein kinases; Pfnek1, Pfmap2, Pfclk1, and Pfclk4. Additionally, isoliensinine was predicted to possess an optimal pharmacokinetics profile and drug-likeness properties.ConclusionThese findings highlight considerable grounds for further exploration of isoliensinine as an amenable scaffold for malaria transmission-blocking chemistry and target validation.

Generating Genetically Modified Plasmodium berghei Sporozoites.

Malaria is a deadly disease caused by the parasite Plasmodium and is transmitted through the bite of female Anopheles mosquitoes. The sporozoite stage of Plasmodium deposited by mosquitoes in the skin of vertebrate hosts undergoes a phase of mandatory development in the liver before initiating clinical malaria. We know little about the biology of Plasmodium development in the liver; access to the sporozoite stage and the ability to genetically modify such sporozoites are critical tools for studying the nature of Plasmodium infection and the resulting immune response in the liver. Here, we present a comprehensive protocol for the generation of transgenic Plasmodium berghei sporozoites. We genetically modify blood-stage P. berghei and use this form to infect Anopheles mosquitoes when they take a blood meal. After the transgenic parasites undergo development in the mosquitoes, we isolate the sporozoite stage of the parasite from the mosquito salivary glands for in vivo and in vitro experimentation. We demonstrate the validity of the protocol by generating sporozoites of a novel strain of P. berghei expressing the green fluorescent protein (GFP) subunit 11 (GFP11), and show how it could be used to investigate the biology of liver-stage malaria.

Exploring the potential of antimalarial nanocarriers as a novel therapeutic approach

Malaria is a life-threatening parasitic disease that affects millions of people worldwide, especially in developing countries. Despite advances in conventional therapies, drug resistance in malaria parasites has become a significant concern. Hence, there is a need for a new therapeutic approach. To combat the disease effectively means eliminating vectors and discovering potent treatments. The nanotechnology research efforts in nanomedicine show promise by exploring the potential use of nanomaterials that can surmount these limitations occurring with antimalarial drugs, which include multidrug resistance or lack of specificity when targeting parasites directly. Utilizing nanomaterials would possess unique advantages over conventional chemotherapy systems by increasing the efficacy levels while reducing side effects significantly by delivering medications precisely within the diseased area. It also provides cheap yet safe measures against Malaria infections worldwide-ultimately improving treatment efficiency holistically without reinventing new methods therapeutically. This review is an effort to provide an overview of the various stages of malaria parasites, pathogenesis, and conventional therapies, as well as the treatment gap existing with available formulations. It explores different types of nanocarriers, such as liposomes, ethosomal cataplasm, solid lipid nanoparticles, nanostructured lipid carriers, polymeric nanocarriers, and metallic nanoparticles, which are frequently employed to boost the efficiency of antimalarial drugs to overcome the challenges and develop effective and safe therapies. The study also highlights the improved pharmacokinetics, enhanced drug bioavailability, and reduced toxicity associated with nanocarriers, making them a promising therapeutic approach for treating malaria.

Mosquito bites and stage-specific antibody responses against Plasmodium falciparum in southern Ghana

BackgroundThe human host elicits specific immune responses after exposure to various life stages of the malaria parasite as well as components of mosquito saliva injected into the host during a mosquito bite. This study describes differences in IgG responses against antigens derived from the sporozoite (PfCSP), asexual stage parasite (PfEBA175) and the gametocyte (Pfs230), in addition to an Anopheles gambiae salivary gland antigen (gSG6-P1), in two communities in Ghana with similar blood stage malaria parasite prevalence.MethodsThis study used archived plasma samples collected from an earlier cross-sectional study that enrolled volunteers aged from 6 months to 70 years from Simiw, peri-urban community (N = 347) and Obom, rural community (N = 291). An archived thick and thin blood smear for microscopy was used for the estimation of Plasmodium parasite density and species and DNA extraction from blood spots and P. falciparum confirmation was performed using PCR. This study used the stored plasma samples to determine IgG antibody levels to P. falciparum and Anopheles salivary antigens using indirect ELISA.ResultsIndividuals from Simiw had significantly higher levels of IgG against mosquito gSG6-P1 [median (95%CI)] [2.590 (2.452–2.783) ng/mL] compared to those from Obom [2.119 (1.957–2.345) ng/mL], p < 0.0001. Both IgG responses against Pfs230proC (p = 0.0006), and PfCSP (p = 0.002) were significantly lower in volunteers from Simiw compared to the participants from Obom. The seroprevalence of PfEBA-175.5R (p = 0.8613), gSG6-P1 (p = 0.0704), PfCSP (p = 0.7798) IgG were all similar in Obom and Simiw. However, Pfs230 seroprevalence was significantly higher at Obom compared to Simiw (p = 0.0006). Spearman correlation analysis showed no significant association between IgG responses against gSG6-P1, PfCSP, Pfs230proC and PfEBA-175.5R and parasite density at both Obom and Simiw (p > 0.05).ConclusionIn conclusion, the study showed that participants from Simiw had higher concentrations of circulating gSG6-P1 IgG antibodies but lower concentrations of P. falciparum antibodies, PfCSP IgG and Pfs230proC IgG compared to participants from Obom.

Targeting an essential Plasmodium cold shock protein to block growth and transmission of malaria parasite

Cold shock proteins are characterized by the presence of one or more cold shock domains that bestow them with nucleic acid binding ability. Although cold shock proteins are well characterized in bacteria, plants and humans, there is no information on their existence and role in malaria parasite. Here, we have identified and delineated the function of a cold shock protein of Plasmodium falciparum (Pf) 'PfCoSP'. We demonstrate that PfCoSP exhibits nucleic acid binding properties and regulates gene expression. PfCoSP promotes microtubule assembly by interacting with Pf α/β tubulin. We identified a human cold shock protein LIN28A inhibitor 'LI71' as a binding partner of PfCoSP which inhibited PfCoSP-DNA and α/β tubulin interactions and, also inhibited the development of asexual blood stages and gametocyte stage of malaria parasite. Because PfCoSP is essential for parasite survival, characterization of its interacting partners may form the basis for development of future anti-malarials.

Understanding the mode of transmission of malaria agents: an astonishing series of hypotheses, observations, research and controversy

In 1880, Laveran observed the causative agent of malaria. As early as 1884, he considered that mosquitoes could be responsible for the transmission of haematozoa, a hypothesis which resulted from the observation and reflection of an informed hygienist. But, as Laveran himself said, "the opinion that I defended was considered by most observers to be highly unlikely".Nearly 15 years after the discovery of the haematozoan, the elucidation of the mechanism of transmission still proved difficult to establish. A link with the existence of swamps had been established a long time before, but the true mode of transmission remained a mystery until the end of the 19th century. The implication, by Manson in 1877, of mosquitoes in the cycle of the Bancroftian filaria, then other observations of the same order, ended up attracting the attention of malariologists. Laveran himself was quickly convinced of the role of mosquitoes in carrying out the natural cycle and propagating Plasmodium, but this theory had as many detractors as supporters.In 1897, Ross showed the presence of oocysts on the stomach of mosquitoes previously gorged on a malaria patient, then in 1898, of sporozoites of bird plasmodia in mosquitoes. He was convinced that, through their bite, these insects were responsible for the transmission of human malaria agents, without being able to prove it. The results obtained by Ross were immediately confirmed in Italy by Grassi and his collaborators who, in November 1898, described the stages of Plasmodium in man and, through various experiments carried out in collaboration with British researchers, showed the role of Anopheles, a result far from being accepted by all. Skepticism persisted for a long time.An excellent protozoologist, Laveran was not an entomologist. He was however among the first defenders of the anopheline theory. He worked extensively on establishing the relationships between Anopheles mosquitoes and malaria and took a close interest in the environmental conditions of the transmission. In his mind, malaria fever should henceforth be classified as a preventable disease. An era of hope thus dawned: malaria prophylaxis, based on fight against mosquitoes, could begin.

Malaria Vaccine Development: Challenges and Prospects

Background: The development of a licensed malaria vaccine has been challenging due to the complex multi-stage life cycle, antigenic variation, and genetic diversity of Plasmodium. Numerous vaccines have been developed for various stages of Plasmodium, including pre-erythrocytic, blood stage, placenta, and transmission-blocking vaccines. However, none of these vaccines are completely effective and have high reactogenicity.Aim: The aim of this review is to examine the challenges and prospects in developing an effective malaria vaccine, with a focus on the development of a multi-stage or multivalent malaria vaccine.Materials and Methods: The authors conducted a review of the literature on malaria vaccine development, focusing on the challenges and prospects of developing an effective malaria vaccine.Results: Efforts are underway to develop a multi-stage or multivalent malaria vaccine (MultiMalVax) that may best target sporozoite development and neutralize merozoites, hepatocytes, and erythrocytes. A thorough understanding of potential vaccine targets and how immunity works is critical to developing a fully effective malaria vaccine.Conclusion: The challenges associated with developing an effective malaria vaccine are significant due to the complex nature of the Plasmodium life cycle. However, the development of a multi-stage or multivalent malaria vaccine offers prospects for overcoming these challenges and developing a fully effective malaria vaccine. A better understanding of potential vaccine targets and how immunity works is crucial in developing an effective vaccine against malaria.

Using qPCR to compare the detection of Plasmodium vivax oocysts and sporozoites in Anopheles farauti mosquitoes between two DNA extraction methods

BackgroundCurrently, the gold standard to assess parasite developmental stages in mosquitoes is light microscopy. Microscopy can miss low-density infections, is time-consuming and not species-specific. Enzyme-linked immunosorbent assay (ELISA) has been the alternative technique to evaluate the infectivity of mosquitoes especially in field studies however it is semi-quantitative. Molecular techniques that have been used to detect the mosquito stages of malaria parasites including P. vivax. Here, we present a quantitative real-time assay (qPCR) that can be used to detect low-density P. vivax oocyst and sporozoite infections while comparing parasites extracted by the conventional DNA extraction and heating methods.MethodsColony reared Anopheles farauti mosquitoes were exposed to blood samples collected from infected individuals using a direct membrane feeding assay. The fully fed mosquitoes were kept for 7 and 14 days post-feed before dissection to confirm presence of oocysts and sporozoites. Infected mosquito guts and the salivary glands (with the head and thorax) were stored and DNA was extracted either by heating or by performing conventional column-based DNA extraction. Following DNA extraction the infected samples were subjected to qPCR to detect P. vivax parasites.ResultsDNA extraction of 1 or more oocysts by heating resulted in an overall sensitivity of 78% (57/73) and single oocysts infections were detected with a sensitivity of 82% (15/17) in the heating arm. We observed a 60% (18/30) sensitivity with sporozoites where DNA was extracted using the conventional DNA extraction method. We show that the heating method significantly improved the detection of oocysts over conventional DNA extraction. There was no significant difference in the DNA copy numbers when comparing the detection of oocysts from the conventional DNA extraction versus heating. However, we observed that the DNA copy numbers of the sporozoites detected in the heating arm was significantly higher than in the conventional DNA extraction arm.ConclusionWe have adapted a qPCR assay which, when coupled with heating to release DNA reduces sample processing time and cost. Direct qPCR after heating will be a useful tool when investigating transmission blocking vaccines or antimalarials or when evaluating field caught mosquitoes for the presence of malaria parasites.

The salivary protein Saglin facilitates efficient midgut colonization of Anopheles mosquitoes by malaria parasites.

Malaria is caused by the unicellular parasite Plasmodium which is transmitted to humans through the bite of infected female Anopheles mosquitoes. To initiate sexual reproduction and to infect the midgut of the mosquito, Plasmodium gametocytes are able to recognize the intestinal environment after being ingested during blood feeding. A shift in temperature, pH change and the presence of the insect-specific compound xanthurenic acid have been shown to be important stimuli perceived by gametocytes to become activated and proceed to sexual reproduction. Here we report that the salivary protein Saglin, previously proposed to be a receptor for the recognition of salivary glands by sporozoites, facilitates Plasmodium colonization of the mosquito midgut, but does not contribute to salivary gland invasion. In mosquito mutants lacking Saglin, Plasmodium infection of Anopheles females is reduced, resulting in impaired transmission of sporozoites at low infection densities. Interestingly, Saglin can be detected in high amounts in the midgut of mosquitoes after blood ingestion, possibly indicating a previously unknown host-pathogen interaction between Saglin and midgut stages of Plasmodium. Furthermore, we were able to show that saglin deletion has no fitness cost in laboratory conditions, suggesting this gene would be an interesting target for gene drive approaches.