Expansion microscopy of Plasmodium gametocytes reveals the molecular architecture of a bipartite microtubule organisation centre coordinating mitosis with axoneme assembly.

Transmission of malaria-causing parasites to mosquitoes relies on the production of gametocyte stages and their development into gametes. These stages display various microtubule cytoskeletons and the architecture of the corresponding microtubule organisation centres (MTOC) remains elusive. Combining ultrastructure expansion microscopy (U-ExM) with bulk proteome labelling, we first reconstructed in 3D the subpellicular microtubule network which confers cell rigidity to Plasmodium falciparum gametocytes. Upon activation, as the microgametocyte undergoes three rounds of endomitosis, it also assembles axonemes to form eight flagellated microgametes. U-ExM combined with Pan-ExM further revealed the molecular architecture of the bipartite MTOC coordinating mitosis with axoneme formation. This MTOC spans the nuclear membrane linking cytoplasmic basal bodies to intranuclear bodies by proteinaceous filaments. In P. berghei, the eight basal bodies are concomitantly de novo assembled in a SAS6- and SAS4-dependent manner from a deuterosome-like structure, where centrin, γ-tubulin, SAS4 and SAS6 form distinct subdomains. Basal bodies display a fusion of the proximal and central cores where centrin and SAS6 are surrounded by a SAS4-toroid in the lumen of the microtubule wall. Sequential nucleation of axonemes and mitotic spindles is associated with a dynamic movement of γ-tubulin from the basal bodies to the intranuclear bodies. This dynamic architecture relies on two non-canonical regulators, the calcium-dependent protein kinase 4 and the serine/arginine-protein kinase 1. Altogether, these results provide insights into the molecular organisation of a bipartite MTOC that may reflect a functional transition of a basal body to coordinate axoneme assembly with mitosis.

Expansion microscopy provides new insights into the cytoskeleton of malaria parasites including the conservation of a conoid.

Malaria is caused by unicellular Plasmodium parasites. Plasmodium relies on diverse microtubule cytoskeletal structures for its reproduction, multiplication, and dissemination. Due to the small size of this parasite, its cytoskeleton has been primarily observable by electron microscopy (EM). Here, we demonstrate that the nanoscale cytoskeleton organisation is within reach using ultrastructure expansion microscopy (U-ExM). In developing microgametocytes, U-ExM allows monitoring the dynamic assembly of axonemes and concomitant tubulin polyglutamylation in whole cells. In the invasive merozoite and ookinete forms, U-ExM unveils the diversity across Plasmodium stages and species of the subpellicular microtubule arrays that confer cell rigidity. In ookinetes, we additionally identify an apical tubulin ring (ATR) that colocalises with markers of the conoid in related apicomplexan parasites. This tubulin-containing structure was presumed to be lost in Plasmodium despite its crucial role in motility and invasion in other apicomplexans. Here, U-ExM reveals that a divergent and considerably reduced form of the conoid is actually conserved in Plasmodium species.

Biogenesis and discharge of the rhoptries: Key organelles for entry and hijack of host cells by the Apicomplexa.

Rhoptries are specialized secretory organelles found in the Apicomplexa phylum, playing a central role in the establishment of parasitism. The rhoptry content includes membranous as well as proteinaceous materials that are discharged into the host cell in a regulated fashion during parasite entry. A set of rhoptry neck proteins form a RON complex that critically participates in the moving junction formation during invasion. Some of the rhoptry bulb proteins are associated with the membranous materials and contribute to the formation of the parasitophorous vacuole membrane while others are targeted into the host cell including the nucleus to subvert cellular functions. Here, we review the recent studies on Toxoplasma and Plasmodium parasites that shed light on the key steps leading to rhoptry biogenesis, trafficking, and discharge.

Cost-effectiveness analysis of malaria rapid diagnostic test in the elimination setting.

BACKGROUND: As more and more countries approaching the goal of malaria elimination, malaria rapid diagnostic tests (RDT) was recomendated to be a diagnostic strategy to achieve and maintain the statute of malaria free, as it's less requirments on equipment and experitise than microscopic examination. But there are very few economic evaluations to confirm whether RDT was cost-effective in the setting of malaria elimination. This research aimed to offer evidence for helping decision making on malaria diagnosis strategy. METHODS: A cost-effectiveness analysis was conducted to compare RDT with microscopy examination for malaria diagnosis, by using a decision tree model. There were three strategies of malaria diagnostic testing evaluated in the model, 1) microscopy, 2) RDT, 3) RDT followed by microscopy. The effect indicator was defined as the number of malaria cases treated appropriately. Based on the joint perspective of health sector and patient, costs data were collected from hospital information systems, key informant interviews, and patient surveys. Data collection was conducted in Jiangsu from September 2018 to January 2019. Epidemiological data were obtained from local malaria surveillance reports. A hypothetical cohort of 300 000 febrile patients were simulated to calculate the total cost and effect of each strategy. One-way, two-way, and probabilistic sensitivity analysis were performed to test the robustness of the result. RESULTS: The results showed that RDT strategy was the most effective (245 cases) but also the most costly (United States Dollar [USD] 4.47 million) compared to using microscopy alone (238 cases, USD 3.63 million), and RDT followed by microscopy (221 cases, USD 2.75 million). There was no strategy dominated. One-way sensitivity analysis reflected that the result was sensitive to the change in labor cost and two-way sensitivity analysis indicated that the result was not sensitive to the proportion of falciparum malaria. The result of Monte Carlo simulation showed that RDT strategy had higher effects and higher cost than other strategies with a high probability. CONCLUSIONS: Compared to microscopy and RDT followed by microscopy, RDT strategy had higher effects and higher cost in the setting of malaria elimination.

Three-dimensional electron microscopy analysis reveals endopolygeny-like nuclear architecture segregation in Plasmodium oocyst development.

The genus Plasmodium is a unicellular eukaryotic parasite that is the causative agent of malaria, which is transmitted by Anopheline mosquito. There are a total of three developmental stages in the production of haploid parasites in the Plasmodium life cycle: the oocyst stage in mosquitoes and the liver and blood stages in mammalian hosts. The Plasmodium oocyst stage plays an important role in the production of the first generation of haploid parasites. Nuclear division is the most important event that occurs during the proliferation of all eukaryotes. However, obtaining the details of nuclear division at the oocyst stage is challenging owing to difficulties in preparation. In this study, we used focused-ion-beam-milling combined with scanning-electron-microscopy to report the 3D architecture during nuclear segregations in oocyst stage. This advanced technology allowed us to analyse the 3D details of organelle segregation inside the oocyst during sporogony formation. It was revealed that multiple nuclei were involved with several centrosomes in one germ nucleus during sporozoite budding (endopolygeny). Our high-resolution 3D analysis uncovered the endopolygeny-like nuclear architecture of Plasmodium in the definitive host. This nuclear segregation was different from that in the blood stage, and its similarity to other apicomplexan parasite nuclear divisions such as Sarcocystis is discussed.

Molecular and Morphological Characterization of a Brazilian Lineage of Plasmodium ( Novyella) Unalis in Turdus Spp. (Passeriformes) of the Atlantic Forest, with Remarks on New Hosts and High Genetic Variation.

Plasmodium spp. are haemosporidian protozoans that alternate their live cycles between bloodsucking Culicidae dipterans and vertebrate hosts (mammals, reptiles, and birds). In birds, these parasites are the causative agents of the so-called avian malaria, a disease associated with considerable declines and extinctions in the avifauna in different geographical regions. In this work, we applied a multidisciplinary approach, light microscopy and cytochrome oxidase b (cyt b) gene sequence analysis, for characterization of Plasmodium spp. found in association with wild birds of the genus Turdus, collected in Atlantic forest fragments of southeastern Brazil. From the total 90 analyzed birds, 58 (47 Turdus rufiventris, 9 Turdus leucomelas, 1 Turdus albicollis, and 1 Turdus flavipes) were positively infected with Plasmodium unalis, a haemosporidian that was previously detected in Turdus fuscater in Colombia and in penguins in Brazil, but has never been found in association with these Turdus species of this present work. Moreover, all 7 new sequences of P. unalis cyt b gene clustered into a monophyletic clade with previously characterized P. unalis sequences with a mean genetic divergence of 1.6% and with a maximum divergence of 3.1%, indicating for a high degree of intraspecific polymorphism within this parasitic species. Together, our data highlight the existence a high degree of intraspecific variation within P. unalis and highlight the importance of integrative taxonomy to an accurate identification and characterization of avian haemosporidian parasites.

The apicoplast: now you see it, now you don't.

Parasites such as Plasmodium and Toxoplasma possess a vestigial plastid homologous to the chloroplasts of algae and plants. The plastid (known as the apicoplast; for apicomplexan plastid) is non-photosynthetic and very much reduced, but has clear endosymbiotic ancestry including a circular genome that encodes RNAs and proteins and a suite of bacterial biosynthetic pathways. Here we review the initial discovery of the apicoplast, and recount the major new insights into apicoplast origin, biogenesis and function. We conclude by examining how the apicoplast can be removed from malaria parasites in vitro, ultimately completing its reduction by chemical supplementation.

Morphological and Genetic Diversity of Opisthosporidia: New Aphelid Paraphelidium tribonemae gen. et sp. nov.

Aphelids are a poorly known group of parasitoids of algae that have raised considerable interest due to their pivotal phylogenetic position. Together with Cryptomycota and the highly derived Microsporidia, they have been recently re-classified as the Opisthosporidia, which constitute the sister group to the fungi within the Holomycota. Despite their huge diversity, as revealed by molecular environmental studies, and their phylogenetic interest, only three genera have been described (Aphelidium, Amoeboaphelidium, and Pseudaphelidium), from which 18S rRNA gene sequences exist only for Amoeboaphelidium and Aphelidium species. Here, we describe the life cycle and ultrastructure of a new representative of Aphelida, Paraphelidium tribonemae gen. et sp. nov., and provide the first 18S rRNA gene sequence obtained for this genus. Molecular phylogenetic analysis indicates that Paraphelidium is distantly related to both Aphelidium and Amoebaphelidium, highlighting the wide genetic diversity of aphelids. Paraphelidium tribonemae has amoeboflagellate zoospores containing a lipid-microbody complex, dictyosomes, and mitochondria with rhomboid cristae, which are also present in trophonts and plasmodia. The amoeboid trophont uses pseudopodia to feed from the host cytoplasm. Although genetically distinct, the genus Paraphelidium is morphologically indistinguishable from other aphelid genera and has zoospores able to produce lamellipodia with subfilopodia like those of Amoeboaphelidium.

Lack of knowledge regarding the microscopic diagnosis of malaria by technicians of the laboratory network in Luanda, Angola.

INTRODUCTION: Malaria is still one of the most important public health problems worldwide. The diagnosis of this disease is still mainly based on thick blood films.  OBJECTIVE: To evaluate the knowledge about malaria diagnosis of the technicians of the public health network in Luanda, Angola, by means of a survey.  MATERIALS AND METHODS: This survey was carried out in three phases. In the first one, open interviews were done to technicians related with the different procedures for malaria diagnosis. In the second one, a preliminary questionnaire was prepared and evaluated. In the third phase, a definitive questionnaire was applied to 120 technicians from Luanda between April and July, 2013. The proportions of correct and incorrect answers were compared for every question of the survey.  RESULTS: Significantly higher proportions of incorrect answers (p<0.05) were found in the questions related to clinical manifestations, 68/52 (p<0.05), species of Plasmodium according to geographical areas, 76/44 (p<0.05), the type of granulations according to species, 96/24 (p<0.01), the class of microscope magnifying glasses used to observe the thick smear, 105/15 (p<0.01), the thick smear report, 76/44 (p<0.01), the time and preparation of different stain solutions, 81/39 (p<0.01), and the number of parasites counted per 200 leukocytes, 96/24 (p<0.01).  CONCLUSIONS: Various failures for the microscopic diagnosis of malaria were observed amongst the evaluated technicians. These results will be useful as a baseline study before applying an educational intervention aimed to improve the quality of malaria diagnosis in Luanda's laboratory network.

Species-specific escape of Plasmodium sporozoites from oocysts of avian, rodent, and human malarial parasites.

BACKGROUND: Malaria is transmitted when an infected mosquito delivers Plasmodium sporozoites into a vertebrate host. There are many species of Plasmodium and, in general, the infection is host-specific. For example, Plasmodium gallinaceum is an avian parasite, while Plasmodium berghei infects mice. These two parasites have been extensively used as experimental models of malaria transmission. Plasmodium falciparum and Plasmodium vivax are the most important agents of human malaria, a life-threatening disease of global importance. To complete their life cycle, Plasmodium parasites must traverse the mosquito midgut and form an oocyst that will divide continuously. Mature oocysts release thousands of sporozoites into the mosquito haemolymph that must reach the salivary gland to infect a new vertebrate host. The current understanding of the biology of oocyst formation and sporozoite release is mostly based on experimental infections with P. berghei, and the conclusions are generalized to other Plasmodium species that infect humans without further morphological analyses. RESULTS: Here, it is described the microanatomy of sporozoite escape from oocysts of four Plasmodium species: the two laboratory models, P. gallinaceum and P. berghei, and the two main species that cause malaria in humans, P. vivax and P. falciparum. It was found that sporozoites have species-specific mechanisms of escape from the oocyst. The two model species of Plasmodium had a common mechanism, in which the oocyst wall breaks down before sporozoites emerge. In contrast, P. vivax and P. falciparum sporozoites show a dynamic escape mechanism from the oocyst via polarized propulsion. CONCLUSIONS: This study demonstrated that Plasmodium species do not share a common mechanism of sporozoite escape, as previously thought, but show complex and species-specific mechanisms. In addition, the knowledge of this phenomenon in human Plasmodium can facilitate transmission-blocking studies and not those ones only based on the murine and avian models.

The inner membrane complex through development of Toxoplasma gondii and Plasmodium.

Plasmodium spp. and Toxoplasma gondii are important human and veterinary pathogens. These parasites possess an unusual double membrane structure located directly below the plasma membrane named the inner membrane complex (IMC). First identified in early electron micrograph studies, huge advances in genetic manipulation of the Apicomplexa have allowed the visualization of a dynamic, highly structured cellular compartment with important roles in maintaining the structure and motility of these parasites. This review summarizes recent advances in the field and highlights the changes the IMC undergoes during the complex life cycles of the Apicomplexa.

Plasmodium circumflexum in a Shikra (Accipiter badius): phylogeny and ultra-structure of the haematozoa.

A wild-caught, juvenile Shikra (Accipiter badius) was evaluated for rehabilitation at the Kasetsart University Raptor Rehabilitation Unit (KURRU) with a history of weakness. Plasmodium sp. was observed by both light and electron microscopy in blood obtained on day 1 of evaluation. Based on the appearance of erythrocytic meronts and gametocytes, the parasites were defined as Plasmodium (Giovannolaia) circumflexum. The sequence analysis of the mitochondrial cytochrome b gene from the plasmodia was closely related to parasites found in the Grey-headed woodpecker from Myanmar and the Brown hawk-owl from Singapore. Transmission electron microscopic examination revealed organelles in the haematozoa and heterophils that ingested the plasmodia. This is the first recorded case of Plasmodium circumflexum in a wild Shikra. This note emphasises the molecular characterisation and ultra-structure of the haematozoa.

Structural basis for chirality and directional motility of Plasmodium sporozoites.

Plasmodium sporozoites can move at high speed for several tens of minutes, which is essential for the initial stage of a malaria infection. The crescent-shaped sporozoites move on 2D substrates preferably in the same direction on circular paths giving raise to helical paths in 3D matrices. Here we determined the structural basis that underlies this type of movement. Immature, non-motile sporozoites were found to lack the subpellicular network required for obtaining the crescent parasite shape. In vitro, parasites moving in the favoured direction move faster and more persistent than the few parasites that move in the opposite direction. Photobleaching experiments showed that sporozoites flip their ventral side up when switching the direction of migration. Cryo-electron tomography revealed a polarized arrangement of microtubules and polar rings towards the substrate in Plasmodium sporozoites, but not in the related parasite Toxoplasma gondii. As a consequence, secretory vesicles, which release proteins involved in adhesion, migration and invasion at the front end of the parasite, are delivered towards the substrate. The resulting chiral structure of the parasite appears to determine the unique directionality of movement and could explain how the sporozoite achieves rapid and sustained directional motility in the absence of external stimuli.

New ultrastructural analysis of the invasive apparatus of the Plasmodium ookinete.

Invasion of the mosquito midgut by the Plasmodium ookinete determines the success of transmission of malaria parasites from humans to mosquitoes and therefore, is a potential target for molecular intervention. Here, we show higher-resolution ultrastructural details of developing and mature P. gallinaceum ookinetes than previously available. Improved fixation and processing methods yielded substantially improved transmission electron micrographs of ookinetes, particularly with regard to visualization of subcellular secretory and other organelles. These new images provide new insights into the synthesis and function of vital invasive machinery focused on the following features: apical membrane protrusions presumptively used for attachment and protein secretion, dark spherical bodies at the apical end of the mature ookinete, and the presence of a dense array of micronemes apposed to microtubules at the apical end of the ookinete involved in constitutive secretion. This work advances understanding of the molecular and cellular details of the Plasmodium ookinete and provides the basis of future, more detailed mechanistic experimentation on the biology of the Plasmodium ookinete.

The malaria digestive vacuole.

During the development of malaria parasites within human erythrocytes, the fusion of digestive vesicles gives rise to a large digestive vacuole (DV). This organelle, which is maintained at low pH, processes 60-80 percent of the erythrocyte hemoglobin to provide a pool of amino acids that is crucial for parasite growth and development. During proteolysis, heme is released from hemoglobin as a toxic byproduct and is detoxified by biocrystallization to hemozoin. Proteases that contribute to hemoglobin breakdown, as well as other DV-associated proteins, arrive at this site via several different transport pathways. Antimalarial quinoline drugs, such as chloroquine, act by binding to heme and thus prevent its sequestration into hemozoin. Other drugs, such as artemisinin, may cause oxidative damage of DV macromolecules and membranes. The membrane of the DV contains ion pumps and transporters that maintain its low pH but are also pivotal in the development of parasite resistance to several antimalarial drugs. Methods for the isolation of the DV organelle have been developed to study the biogenesis and function of this important organelle.

Visualizing the 3D architecture of multiple erythrocytes infected with Plasmodium at nanoscale by focused ion beam-scanning electron microscopy.

Different methods for three-dimensional visualization of biological structures have been developed and extensively applied by different research groups. In the field of electron microscopy, a new technique that has emerged is the use of a focused ion beam and scanning electron microscopy for 3D reconstruction at nanoscale resolution. The higher extent of volume that can be reconstructed with this instrument represent one of the main benefits of this technique, which can provide statistically relevant 3D morphometrical data. As the life cycle of Plasmodium species is a process that involves several structurally complex developmental stages that are responsible for a series of modifications in the erythrocyte surface and cytoplasm, a high number of features within the parasites and the host cells has to be sampled for the correct interpretation of their 3D organization. Here, we used FIB-SEM to visualize the 3D architecture of multiple erythrocytes infected with Plasmodium chabaudi and analyzed their morphometrical parameters in a 3D space. We analyzed and quantified alterations on the host cells, such as the variety of shapes and sizes of their membrane profiles and parasite internal structures such as a polymorphic organization of hemoglobin-filled tubules. The results show the complex 3D organization of Plasmodium and infected erythrocyte, and demonstrate the contribution of FIB-SEM for the obtainment of statistical data for an accurate interpretation of complex biological structures.

A new morphologically distinct avian malaria parasite that fails detection by established polymerase chain reaction-based protocols for amplification of the cytochrome B gene.

Plasmodium polymorphum n. sp. (Haemosporida, Plasmodiidae) was found in the skylark, Alauda arvensis (Passeriformes: Alaudidae), during autumnal migration in southern Italy. This organism is illustrated and described based on the morphology of its blood stages. The most distinctive feature of this malaria parasite is the clear preference of its blood stages (trophozoites, meronts, and gametocytes) for immature red blood cells, including erythroblasts. Based on preference of erythrocytic meronts for immature red blood cells, P. polymorphum is most similar to species of the subgenus Huffia . This parasite can be readily distinguished from all other bird malaria parasites, including Plasmodium ( Huffia ) spp., due to preferential development and maturation of its gametocytes in immature red blood cells, a unique character for avian Plasmodium spp. In addition, the margins of nuclei in blood stages of P. polymorphum are markedly smooth and distinct; this is also a distinct diagnostic feature of this parasite. Plasmodium polymorphum has been recorded only in the skylark; it is probably a rare parasite, whose host range and geographical distribution remain unclear. Microscopic examination detected a light infection of Plasmodium relictum (lineage GRW11, parasitemia of <0.01%) in the same sample with P. polymorphum ; the latter parasite clearly predominated (3.5% parasitemia). However, experienced researchers were unable to detect sequences of mitochondrial cytochrome b gene (cyt b ) of P. polymorphum from the microscopically positive sample by using published and newly designed primers for DNA amplification of avian Plasmodium spp. The light parasitemia of P. relictum was easily detectable using several polymerase chain reaction (PCR)-based assays, but P. polymorphum was undetectable in all applied assays. Quantitative PCR also showed the presence of light parasitemia (0.06%) of the lineage GRW11 in this sample. This supports the conclusion that the morphologically distinct parasite observed along with P. relictum and predominant in the sample is genetically dissimilar from the lineage GRW11 based on cyt b sequence. In samples with co-infections, general PCR protocols tend to favor the amplification of the parasite with the higher parasitemia or the amplification with the best matching sequence to the primers. Because the parasitemia of P. polymorphum was >50-fold higher than that of P. relictum and several different primers were tested, we suggest that the failure to amplify P. polymorphum is a more complex problem than why co-infections are commonly overlooked in PCR-based studies. We suggest possible explanations of these results and call for additional research on evolution of mitochondrial genome of hemosporidian parasites.

Understanding parasite transmission through imaging approaches.

Unicellular parasites are of high medical relevance as they cause such devastating diseases as malaria or sleeping sickness. Besides the search for improved treatments, research on these parasites is valuable as they constitute interesting model cells to study basic processes of life. They can also serve as valuable reality checks for our presumed understanding of biological processes that emerge from the study of human or yeast cells, as our common ancestor with many parasites is much older than the one with yeast. But working with parasites can be tricky and time-consuming, if not outright impossible. Here, we focus on examples from imaging studies investigating the transmission of the malaria parasite. Achieving an understanding of the processes important for malaria transmission necessitates different imaging approaches and new molecular and material technologies. The discussed techniques will include in vivo imaging of pathogens in living animals, screening methodologies, and new materials as surrogate 3D environments.

Real time polymerase chain reaction for the detection of malarial parasite.

OBJECTIVE: To evaluate the sensitivity and specificity of real time polymerase chain reaction (PCR) for the detection of Malarial parasite. STUDY DESIGN: Descriptive cross-sectional study. PLACE AND DURATION OF STUDY: The Armed Forces Institute of Pathology (AFIP), Rawalpindi, from April to June 2010. METHODOLOGY: A total of 60 Leishman stained blood films with clinical suspicion of malaria were studied by light microscopy for detection of malaria parasite (MP). The samples were also subjected to real time PCR for the small subunit (SSU) rRNA gene of MP found in all the four subspecies of Plasmodium. Real time PCR was done by the Taqman probe method. One sample positive for MP was serially diluted with ABO compatible blood, and light microscopy and real time PCR were performed on all dilutions. Results of light microscopy and real time PCR were compared. Sensitivity and specificity were calculated using PCR as the gold standard. RESULTS: PCR detected MP in 33 samples with sensitivity and specificity of 100% whereas light microscopy could detect MP in 30 samples. Sensitivity and specificity of light microscopy was 90.9% and 100% respectively. In the serially diluted blood sample, MP was visible at 1/16 dilution whereas the PCR showed positive results even at 1/512 dilution. CONCLUSION: Real time PCR is more sensitive than light microscopy for the detection of malarial parasite.

Crystalloid body, refractile body and virus-like particles in Apicomplexa: what is in there?

The phylum of Apicomplexa comprises parasitic protozoa that share distinctive features such as the apical complex, the apicoplast, specialized cytoskeletal components and secretory organelles. Other unique cytoplasmic inclusions sharing similar features have been described in some representatives of Apicomplexa, although under different denominations. These are the crystalloid body, present for example in Cryptosporidium, Plasmodium and Cystoisospora; the refractile body in Eimeria and Lankesterella; and virus-like particles, also present in Eimeria and Cryptosporidium. Yet, the specific role of these cytoplasmic inclusions in the cell cycle of these protozoa is still unknown. Here, we discuss their morphology, possible inter-relatedness and speculate upon their function to bring these organelles back to the attention of the scientific community and promote new interest towards original research on these elusive structures.