SUMMARY Blastocystis is an unusual enteric protozoan parasite of humans and many animals. It has a worldwide distribution and is often the most commonly isolated organism in parasitological surveys. The parasite has been described since the early 1900s, but only in the last decade or so have there been significant advances in our understanding of Blastocystis biology. However, the pleomorphic nature of the parasite and the lack of standardization in techniques have led to confusion and, in some cases, misinterpretation of data. This has hindered laboratory diagnosis and efforts to understand its mode of reproduction, life cycle, prevalence, and pathogenesis. Accumulating epidemiological, in vivo, and in vitro data strongly suggest that Blastocystis is a pathogen. Many genotypes exist in nature, and recent observations indicate that humans are, in reality, hosts to numerous zoonotic genotypes. Such genetic diversity has led to a suggestion that previously conflicting observations on the pathogenesis of Blastocystis are due to pathogenic and nonpathogenic genotypes. Recent epidemiological, animal infection, and in vitro host-Blastocystis interaction studies suggest that this may indeed be the case. This review focuses on such recent advances and also provides updates on laboratory and clinical aspects of Blastocystis spp.

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In blood vessels with luminal diameter less than 300 µm, red blood cells (RBCs) which are smaller in size and more deformable than leukocytes, migrate to the axial centre of the vessel due to flow velocity gradient within the vessels. This phenomenon displaces the leukocytes to the vessel wall and is aptly termed as margination. Here, we demonstrate using microfluidics that stiffer malaria-infected RBCs (iRBCs) behave similar to leukocytes and undergo margination towards the sidewalls. This provides better understanding of the hemodynamic effects of iRBCs in microcirculation and its contribution to pathophysiological outcome relating to cytoadherence to endothelium. In this work, cell margination is mimicked for the separation of iRBCs from whole blood based on their reduced deformability. The malaria infected sample was tested in a simple long straight channel microfluidic device fabricated in polydimethylsiloxane. In this microchannel, cell margination was directed along the channel width with the iRBCs aligning near each sidewall and then subsequently removed using a 3-outlet system, thus achieving separation. Tests were conducted using ring stage and late trophozoite/schizont stage iRBCs. Device performance was quantified by analyzing the distribution of these iRBCs across the microchannel width at the outlet and also conducting flow cytometry analysis. Results indicate recovery of approximately 75% for early stage iRBCs and >90% for late stage iRBCs at the side outlets. The simple and passive system operation makes this technique ideal for on-site iRBCs enrichment in resource-limited settings, and can be applied to other blood cell diseases, e.g. sickle cell anemia and leukemia, characterized by changes in cell stiffness.

Key Points• Plasmodium vivax merozoites preferentially infect a subgroup of reticulocytes generally restricted to the bone marrow.• Accelerated "maturation" of infected reticulocytes.Plasmodium vivax merozoites only invade reticulocytes, a minor though heterogeneous population of red blood cell precursors that can be graded by levels of transferrin receptor (CD71) expression. The development of a protocol that allows sorting reticulocytes into defined developmental stages and a robust ex vivo P vivax invasion assay has made it possible for the first time to investigate the fine-scale invasion preference of P vivax merozoites. Surprisingly, it was the immature reticulocytes (CD71 1) that are generally restricted to the bone marrow that were preferentially invaded, whereas older reticulocytes (CD71 2 ), principally found in the peripheral blood, were rarely invaded. Invasion assays based on the CD71 1 reticulocyte fraction revealed substantial postinvasion modification. Thus, 3 to 6 hours after invasion, the initially biomechanically rigid CD71 1 reticulocytes convert into a highly deformable CD71 2 infected red blood cell devoid of host reticular matter, a process that normally spans 24 hours for uninfected reticulocytes. Concurrent with these changes, clathrin pits disappear by 3 hours postinvasion, replaced by distinctive caveolae nanostructures. These 2 hitherto unsuspected features of P vivax invasion, a narrow preference for immature reticulocytes and a rapid remodeling of the host cell, provide important insights pertinent to the pathobiology of the P vivax infection. (Blood. 2015;125(8):1314-1324

Blastocystis is an enteric protistan parasite of uncertain clinical relevance. Recent studies indicate that the parasite is a species complex and humans are potentially hosts to nine Blastocystis subtypes, most of which are zoonotic. Subtype 3 is the most common in prevalence studies, followed by subtype 1. Laboratory diagnosis is challenging; the currently recommended diagnostic approach is trichrome staining of direct smears coupled with stool culture. Polymerase chain reaction testing from stools or culture is useful for determining Blastocystis subtype information. The controversial pathogenesis of Blastocystis is attributed to subtype variations in virulence; although current studies seem to support this idea, evidence suggests other factors also contribute to the clinical outcome of the infection. Clinical signs and symptoms of blastocystosis include abdominal pain, diarrhea, bloating, and flatulence. Extraintestinal manifestations, predominantly cutaneous, also were reported. In vitro and animal studies shed new light on the pathobiology of Blastocystis.

During intraerythrocytic development, Plasmodium falciparum exports proteins that interact with the host cell plasma membrane and subplasma membrane-associated spectrin network. Parasiteexported proteins modify mechanical properties of host RBCs, resulting in altered cell circulation. In this work, optical tweezers experiments of cell mechanical properties at normal physiological and febrile temperatures are coupled, for the first time, with targeted gene disruption techniques to measure the effect of a single parasite-exported protein on host RBC deformability. We investigate Pf155/Ring-infected erythrocyte surface antigen (RESA), a parasite protein transported to the host spectrin network, on deformability of ring-stage parasite-harboring human RBCs. Using a set of parental, gene-disrupted, and revertant isogenic clones, we found that RESA plays a major role in reducing deformability of host cells at the early ring stage of parasite development, but not at more advanced stage. We also show that the effect of RESA on deformability is more pronounced at febrile temperature, which ring-stage parasite-harboring RBCs can be exposed to during a malaria attack, than at normal body temperature. malaria ͉ erythrocyte ͉ membrane shear modulus ͉ spectrin ͉ cytoskeleton A fter Plasmodium falciparum merozoite invasion of a human RBC, the parasite differentiates and multiplies for 48 h, leading to rupture of the parasitized RBC (Pf-RBCs) and release of new merozoites in the blood circulation. Throughout this 48-h period, several parasite proteins are introduced into the RBC plasma membrane and submembranous protein skeleton, thereby modifying a range of structural and functional properties of the Pf-RBCs (1-4). The best documented changes occur as P. falciparum matures to the trophozoite (24-36 h) and schizont (36-48 h) stages, when Pf-RBCs display decreased membrane deformability (2-6), become spherical, and develop cytoadherence properties responsible for parasite sequestration in the postcapillary venules of different organs (7,8). In contrast, during early parasite development, ring-stage (0-24 h after invasion) Pf-RBCs preserve their biconcave shape, can circulate in peripheral blood (9), and thus are exposed to the spleen red pulp. Ring-stage Pf-RBCs may pass through this spleen compartment, be expelled from circulation, or return to the circulation once the parasite has been removed (10). Although the relative importance of these different processes and their underlying mechanisms are not fully understood, it is likely that altered deformability of ring-stage Pf-RBCs (11) plays a crucial role in determining Pf-RBCs spleen processing.Introduction of parasite components within the Pf-RBC membrane and cortical cytoskeleton begins soon after RBC invasion, as demonstrated for the well characterized parasite protein Pf155/Ring-infected erythrocyte surface antigen (RESA) (12). This protein is discharged by the invading merozoite and exported to the Pf-RBC membrane where, once phosphorylated, it interacts with the spectrin n...

BackgroundBlastocystis is a highly prevalent anaerobic eukaryotic parasite of humans and animals that is associated with various gastrointestinal and extraintestinal disorders. Epidemiological studies have identified different subtypes but no one subtype has been definitively correlated with disease.ResultsHere we report the 18.8 Mb genome sequence of a Blastocystis subtype 7 isolate, which is the smallest stramenopile genome sequenced to date. The genome is highly compact and contains intriguing rearrangements. Comparisons with other available stramenopile genomes (plant pathogenic oomycete and diatom genomes) revealed effector proteins potentially involved in the adaptation to the intestinal environment, which were likely acquired via horizontal gene transfer. Moreover, Blastocystis living in anaerobic conditions harbors mitochondria-like organelles. An incomplete oxidative phosphorylation chain, a partial Krebs cycle, amino acid and fatty acid metabolisms and an iron-sulfur cluster assembly are all predicted to occur in these organelles. Predicted secretory proteins possess putative activities that may alter host physiology, such as proteases, protease-inhibitors, immunophilins and glycosyltransferases. This parasite also possesses the enzymatic machinery to tolerate oxidative bursts resulting from its own metabolism or induced by the host immune system.ConclusionsThis study provides insights into the genome architecture of this unusual stramenopile. It also proposes candidate genes with which to study the physiopathology of this parasite and thus may lead to further investigations into Blastocystis-host interactions.

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