New insights into the interactions between Blastocystis, the gut microbiota, and host immunity.

The human gut microbiota is a diverse and complex ecosystem that is involved in beneficial physiological functions as well as disease pathogenesis. Blastocystis is a common protistan parasite and is increasingly recognized as an important component of the gut microbiota. The correlations between Blastocystis and other communities of intestinal microbiota have been investigated, and, to a lesser extent, the role of this parasite in maintaining the host immunological homeostasis. Despite recent studies suggesting that Blastocystis decreases the abundance of beneficial bacteria, most reports indicate that Blastocystis is a common component of the healthy gut microbiome. This review covers recent finding on the potential interactions between Blastocystis and the gut microbiota communities and its roles in regulating host immune responses.

Resistance of Blastocystis to chlorine and hydrogen peroxide.

Blastocystis is a ubiquitous, widely distributed protist inhabiting the gastrointestinal tract of humans and other animals. The organism is genetically diverse, and so far, at least 28 subtypes (STs) have been identified with ST1-ST9 being the most common in humans. The pathogenicity of Blastocystis is controversial. Several routes of transmission have been proposed including fecal-oral (e.g., zoonotic, anthroponotic) and waterborne. Research on the latter has gained traction in the last few years with the organism having been identified in various bodies of water, tap water, and rainwater collection containers including water that has been previously filtered and/or chlorinated. Herein, we assessed the resistance of 11 strains maintained in culture, spanning ST1-ST9 to various chlorine and hydrogen peroxide concentrations for 24 h, and performed recovery assays along with re-exposure. Following the treatment with both compounds, all subtypes showed increased resistance, and viability could be visualized at the cellular level. These results are hinting at the presence of mechanism of resistance to both chlorine and hydrogen peroxide. As such, this pilot study can be the platform for developing guidelines for water treatment processes.

Taming the Sentinels: Microbiome-Derived Metabolites and Polarization of T Cells.

A global increase in the prevalence of metabolic syndromes and digestive tract disorders, like food allergy or inflammatory bowel disease (IBD), has become a severe problem in the modern world. Recent decades have brought a growing body of evidence that links the gut microbiome's complexity with host physiology. Hence, understanding the mechanistic aspects underlying the synergy between the host and its associated gut microbiome are among the most crucial questions. The functionally diversified adaptive immune system plays a central role in maintaining gut and systemic immune homeostasis. The character of the reciprocal interactions between immune components and host-dwelling microbes or microbial consortia determines the outcome of the organisms' coexistence within the holobiont structure. It has become apparent that metabolic by-products of the microbiome constitute crucial multimodal transmitters within the host-microbiome interactome and, as such, contribute to immune homeostasis by fine-tuning of the adaptive arm of immune system. In this review, we will present recent insights and discoveries regarding the broad landscape of microbiome-derived metabolites, highlighting the role of these small compounds in the context of the balance between pro- and anti-inflammatory mechanisms orchestrated by the host T cell compartment.

Viability Screen of LOPAC1280 Reveals Tyrosine Kinase Inhibitor Tyrphostin A9 as a Novel Partner Drug for Artesunate Combinations To Target the Plasmodium falciparum Ring Stage.

The emergence of artemisinin-resistant Plasmodium falciparum poses a major threat to current frontline artemisinin combination therapies. Artemisinin resistance is widely associated with mutations in the P. falciparum Kelch13 (PfKelch13) propeller region, leading to delayed parasite clearance and increased survival of early-ring-stage parasites. There is therefore a need to discover novel drugs that are effective against artemisinin-resistant P. falciparum In view of this, our study aimed to identify compounds from the Library of Pharmacologically Active Compounds1280 (LOPAC1280) that could increase the efficacy of artesunate and be used as a potential partner drug for treatment against artemisinin-resistant falciparum malaria. By using a modified ring-stage survival assay, we performed a high-throughput screening of the activities of the 1,280 compounds from the LOPAC library in combination with artesunate against the P. falciparum IPC 5202 field isolate harboring the R539T mutation in the PfKelch13 propeller region. The potencies of the hits against both the IPC 5202 and CamWT_C580Y field isolates were determined through dose-dependent isobologram analyses; CamWT_C580Y has the more prevalent C580Y mutation characteristic of strains with artemisinin resistance. We identified tyrphostin A9 to have synergistic and additive activity against both parasite strains when dosed in combination with artesunate. These findings provide promising novel artesunate combinations that can target the P. falciparum artemisinin-resistant ring stage and insights that may aid in obtaining a better understanding of the mechanism involved in artemisinin resistance.

Strict tropism for CD71+/CD234+ human reticulocytes limits the zoonotic potential of Plasmodium cynomolgi.

Two malaria parasites of Southeast Asian macaques, Plasmodium knowlesi and P cynomolgi, can infect humans experimentally. In Malaysia, where both species are common, zoonotic knowlesi malaria has recently become dominant, and cases are recorded throughout the region. By contrast, to date, only a single case of naturally acquired P cynomolgi has been found in humans. In this study, we show that whereas P cynomolgi merozoites invade monkey red blood cells indiscriminately in vitro, in humans, they are restricted to reticulocytes expressing both transferrin receptor 1 (Trf1 or CD71) and the Duffy antigen/chemokine receptor (DARC or CD234). This likely contributes to the paucity of detectable zoonotic cynomolgi malaria. We further describe postinvasion morphologic and rheologic alterations in P cynomolgi-infected human reticulocytes that are strikingly similar to those observed for P vivax These observations stress the value of P cynomolgi as a model in the development of blood stage vaccines against vivax malaria.

A high content imaging flow cytometry approach to study mitochondria in T cells: MitoTracker Green FM dye concentration optimization.

Mitochondria, the powerhouse of the cell, are known to remodel their membrane structures through the process of fusion or fission. Studies have indicated that T cells adopt different energy metabolic phenotypes, namely oxidative phosphorylation and glycolysis depending on whether they are naïve, effector and memory T cells. It has recently been shown that changes in mitochondrial morphology dictate T cell fate via regulation of their metabolism. Our keen interest in T cell function and metabolism led us to explore and establish a method to study mitochondria in live T cells through a novel high content approach called Imaging Flow Cytometry (IFC). The focus of our current study was on developing a protocol to standardize the concentration of MitoTracker Green FM dye to observe mitochondria in live T cells using IFC. We began the study by using widefield microscopy to confirm the localisation of MitoTracker Green FM labelled mitochondria in live T cells. This was followed by testing various concentrations of the dye to achieve a similar labelling pattern using IFC while eliminating false positive or negative staining. The optimization of the method used to label the mitochondria by IFC for analysis included standardisation of a number of important parameters such as dye concentration, voltage, fluorescence intensity values for acquisition and processing. IFC could potentially be a powerful method to study T cells in a relatively high throughput manner.

Proteogenomic Insights into the Intestinal Parasite Blastocystis sp. Subtype 4 Isolate WR1.

Blastocystis sp. is known for years as a highly prevalent anaerobic eukaryotic parasite of humans and animals. Several monophyletic clades have been delineated based on molecular data, and the occurrence of each subtype in humans and/or animal hosts has been documented. The genome of several representatives has been sequenced revealing specific traits such as an intriguing 3'-end processing of primary transcripts. Here, a first high-throughput proteomics dataset acquired on this difficult-to-cultivate parasite is presented for the zoonotic subtype T4 isolate WR1. Amongst the 2766 detected proteins, we highlighted the role of a small ADP ribosylation factor GTP-binding protein involved in intracellular traffic as major regulator of vesicle biogenesis and a voltage-dependent anion-selective channel protein because both were unexpectedly highly abundant. We show how these data may be used for gaining proteogenomics insights into Blastocystis sp. specific molecular mechanisms. We evidenced for the first time by proteogenomics a functional termination codon derived from transcript polyadenylation for seven different key cellular components.

Plasmodium vivax: restricted tropism and rapid remodeling of CD71-positive 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(+)) that are generally restricted to the bone marrow that were preferentially invaded, whereas older reticulocytes (CD71(-)), principally found in the peripheral blood, were rarely invaded. Invasion assays based on the CD71(+) reticulocyte fraction revealed substantial postinvasion modification. Thus, 3 to 6 hours after invasion, the initially biomechanically rigid CD71(+) reticulocytes convert into a highly deformable CD71(-) 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.

Overcoming Chloroquine Resistance in Malaria: Design, Synthesis, and Structure-Activity Relationships of Novel Hybrid Compounds.

Resistance to antimalarial therapies, including artemisinin, has emerged as a significant challenge. Reversal of acquired resistance can be achieved using agents that resensitize resistant parasites to a previously efficacious therapy. Building on our initial work describing novel chemoreversal agents (CRAs) that resensitize resistant parasites to chloroquine (CQ), we herein report new hybrid single agents as an innovative strategy in the battle against resistant malaria. Synthetically linking a CRA scaffold to chloroquine produces hybrid compounds with restored potency toward a range of resistant malaria parasites. A preferred compound, compound 35, showed broad activity and good potency against seven strains resistant to chloroquine and artemisinin. Assessment of aqueous solubility, membrane permeability, and in vitro toxicity in a hepatocyte line and a cardiomyocyte line indicates that compound 35 has a good therapeutic window and favorable drug-like properties. This study provides initial support for CQ-CRA hybrid compounds as a potential treatment for resistant malaria.

A high-content phenotypic screen reveals the disruptive potency of quinacrine and 3',4'-dichlorobenzamil on the digestive vacuole of Plasmodium falciparum.

Plasmodium falciparum is the etiological agent of malignant malaria and has been shown to exhibit features resembling programmed cell death. This is triggered upon treatment with low micromolar doses of chloroquine or other lysosomotrophic compounds and is associated with leakage of the digestive vacuole contents. In order to exploit this cell death pathway, we developed a high-content screening method to select compounds that can disrupt the parasite vacuole, as measured by the leakage of intravacuolar Ca(2+). This assay uses the ImageStream 100, an imaging-capable flow cytometer, to assess the distribution of the fluorescent calcium probe Fluo-4. We obtained two hits from a small library of 25 test compounds, quinacrine and 3',4'-dichlorobenzamil. The ability of these compounds to permeabilize the digestive vacuole in laboratory strains and clinical isolates was validated by confocal microscopy. The hits could induce programmed cell death features in both chloroquine-sensitive and -resistant laboratory strains. Quinacrine was effective at inhibiting field isolates in a 48-h reinvasion assay regardless of artemisinin clearance status. We therefore present as proof of concept a phenotypic screening method with the potential to provide mechanistic insights to the activity of antimalarial drugs.

Statin pleiotropy prevents rho kinase-mediated intestinal epithelial barrier compromise induced by Blastocystis cysteine proteases.

Blastocystis is an enteric parasite that causes acute and chronic intestinal infections, often non-responsive to conventional antibiotics. The effects of Blastocystis infections on human epithelial permeability are not known, and molecular mechanisms of Blastocystis-induced intestinal pathology remain unclear. This study was conducted to determine whether Blastocystis species alters human intestinal epithelial permeability, to assess whether these abnormalities are rho kinase (ROCK)-dependent, and to investigate the therapeutic potential of the HMG-CoA reductase inhibitor Simvastatin in altered intestinal epithelial barrier function. The effect of metronidazole resistant (Mz(r)) Blastocystis isolated from a symptomatic patient on human colonic epithelial monolayers (Caco-2) was assessed. Modulation of enterocyte myosin light chain phosphorylation, transepithelial fluorescein isothiocyanate-dextran fluxes, transepithelial resistance, cytoskeletal F-actin and tight junctional zonula occludens-1 (ZO-1) by parasite cysteine proteases were measured in the presence or absence of HMG-CoA reductase and ROCK inhibition. Blastocystis significantly decreased transepithelial resistance, increased epithelial permeability, phosphorylated myosin light chain and reorganized epithelial actin cytoskeleton and ZO-1. These alterations were abolished by inhibition of enterocyte ROCK, HMG-CoA reductase and parasite cysteine protease. Our findings suggest that cysteine proteases of Mz(r) Blastocystis induce ROCK-dependent disruption of intestinal epithelial barrier function and correlates with reorganization of cytoskeletal F-actin and tight junctional ZO-1. Simvastatin prevented parasite-induced barrier-compromise, suggesting a therapeutic potential of statins in intestinal infections.

Interactions between Blastocystis subtype ST4 and gut microbiota in vitro.

BACKGROUND: Blastocystis ST4 is a common protistan parasite of the gastrointestinal tract of humans and a wide range of animals. While it has been suggested that colonization with ST4 is associated with healthy gut microbiota, how ST4 influences the gut microbiota remains poorly studied. This study aimed to examine the interactions between ST4 and several intestinal bacteria using in vitro co-culture systems, and to further investigate the mechanism of interaction and its effect on the epithelial barrier integrity of HT-29 cells. METHODS: Seven intestinal bacteria Bacteroides fragilis, Bifidobacterium longum, Bacillus subtilis, Bacteroides vulgatus, Escherichia coli, Enterococcus faecalis, and Lactobacillus brevis were co-cultured with Blastocystis ST4 in vitro. Flow cytometry and quantitative reverse-transcription polymerase chain reaction (qRT-PCR) were used to determine the role of reactive oxygen species (ROS) and bacteria oxidoreductase genes, respectively, in response to Blastocystis co-incubation. Transepithelial electrical resistance (TEER) and flux assays were performed to assess the effect of microbiota representatives on the integrity of the intestinal epithelial barrier. RESULTS: Co-incubation with Blastocystis ST4 showed a beneficial influence on most intestinal bacteria, while ST4 significantly inhibited the growth of B. vulgatus, a common pathogen in the genus Bacteroides. The decrease in B. vulgatus when co-incubated with Blastocystis ST4 was associated with high levels of ROS and the upregulation of oxidative stress-related genes. Furthermore, co-incubation with Blastocystis ST4 was able to protect the intestinal epithelial barrier from damage by B. vulgatus. CONCLUSIONS: This study demonstrated, for the first time, that Blastocystis ST4 has beneficial effects on intestinal commensal bacteria in vitro, and can inhibit the growth of pathogenic B. vulgatus. Combined with previous microbiome research on ST4, our data suggest that ST4 may be a beneficial commensal.

Infection with pathogenic Blastocystis ST7 is associated with decreased bacterial diversity and altered gut microbiome profiles in diarrheal patients.

BACKGROUND: Blastocystis is a common protistan parasite inhabiting the gastrointestinal tract of humans and animals. While there are increasing reports characterizing the associations between Blastocystis and the gut microbiome in healthy individuals, only a few studies have investigated the relationships between Blastocystis and the gut microbiota in diarrheal patients. METHODS: The effects of a specific subtype (ST7) of Blastocystis on the composition of gut microbiota in diarrheal patients were investigated using 16S ribosomal RNA (rRNA) gene sequencing and bioinformatic analyses. RESULTS: Compared with diarrheal patients without Blastocystis, diarrheal patients infected with Blastocystis ST7 exhibited lower bacterial diversity. Beta diversity analysis revealed significant differences in bacterial community structure between ST7-infected and Blastocystis-free patients. The proportion of Enterobacteriaceae and Escherichia-Shigella were significantly enriched in ST7-infected patients. In contrast, the abundance of Bacteroides and Parabacteroides were more prevalent in Blastocystis-free patients. CONCLUSIONS: The results of this study revealed, for the first time, that infection with Blastocystis ST7 is associated with lower bacterial diversity and altered microbial structure in diarrheal patients. Our study on clinical diarrheal patients is also the first to reinforce the notion that ST7 is a pathogenic subtype of Blastocystis.

Changes in Gut Microbiota Composition Associated with the Presence of Enteric Protist Blastocystis in Captive Forest Musk Deer (Moschus Berezovskii).

Blastocystis is a common protistan parasite inhabiting the gastrointestinal tract of a wide range of hosts including humans and domestic and wild animals. Many studies have revealed the associations between Blastocystis and gut microbiome in humans. However, only a few studies have focused on the associations between Blastocystis and gut microbiome of animals, especially in forest musk deer (Moschus berezovskii). We investigated the effects of the Blastocystis colonization on the intestinal bacterial community compositions using amplicon sequencing targeting the V4 variable region of the 16S rRNA. Two subtypes of Blastocystis (ST5 and ST10) and Blastocystis-free (control) were included in this study. We found that compared with the forest musk deer without Blastocystis, ST10-colonized forest musk deer had higher bacterial richness and diversity, while ST5-colonized forest musk deer showed a comparable bacterial diversity. Likewise, beta diversity revealed significant differences in bacterial community structure between ST10-colonized and Blastocystis-free forest musk deer. The proportion of Bacteroidetes were significantly enriched in ST10-colonized forest musk deer. Bacterial community structure between ST5-colonized and Blastocystis-free forest musk deer did not differ significantly. The present study explored the associations between Blastocystis and gut microbial community of forest musk deer for the first time, and revealed ST10 colonization, instead of ST5, is associated with higher bacterial diversity and shifted microbial structure. Our data provides valuable insights into the associations between gut microbiomes and parasites. IMPORTANCE Forest musk deer is listed as an endangered species by International Union for Conservation of Nature Red List, and the Chinese government has introduced captivity breeding measures to curb the rapid decline of the musk deer population since the 1950s. It has been suggested that Blastocystis colonization can modulate the composition of the host's intestinal microbiota, thereby affecting the host health. The present study investigated the effects of the Blastocystis colonization on the gut microbiota in the feces of forest musk deer in Sichuan Province, China. Two subtypes (ST5 and ST10) have differential effects on the bacterial diversity and community composition, suggesting that the study of Blastocystis should be distinguished at the subtype level. Because the pathogenicity of Blastocystis is controversial, pathogenic, or commensal, continuous monitoring of the impact of Blastocystis colonization on the intestinal microbiota is of great significance to assess its health effects on forest musk deer.

A metronidazole-resistant isolate of Blastocystis spp. is susceptible to nitric oxide and downregulates intestinal epithelial inducible nitric oxide synthase by a novel parasite survival mechanism.

Blastocystis, one of the most common parasites colonizing the human intestine, is an extracellular, noninvasive, luminal protozoan with controversial pathogenesis. Blastocystis infections can be asymptomatic or cause intestinal symptoms of vomiting, diarrhea, and abdominal pain. Although chronic infections are frequently reported, Blastocystis infections have also been reported to be self-limiting in immunocompetent patients. Characterizing the host innate response to Blastocystis would lead to a better understanding of the parasite's pathogenesis. Intestinal epithelial cells produce nitric oxide (NO), primarily on the apical side, in order to target luminal pathogens. In this study, we show that NO production by intestinal cells may be a host defense mechanism against Blastocystis. Two clinically relevant isolates of Blastocystis, ST-7 (B) and ST-4 (WR-1), were found to be susceptible to a range of NO donors. ST-7 (B), a metronidazole-resistant isolate, was found to be more sensitive to nitrosative stress. Using the Caco-2 model of human intestinal epithelium, Blastocystis ST-7 (B) but not ST-4 (WR-1) exhibited dose-dependent inhibition of Caco-2 NO production, and this was associated with downregulation of inducible nitric oxide synthase (iNOS). Despite its higher susceptibility to NO, Blastocystis ST-7 (B) may have evolved unique strategies to evade this potential host defense by depressing host NO production. This is the first study to highlight a strain-to-strain variation in the ability of Blastocystis to evade the host antiparasitic NO response.

A rapid, high-throughput viability assay for Blastocystis spp. reveals metronidazole resistance and extensive subtype-dependent variations in drug susceptibilities.

Blastocystis is an emerging protistan parasite of controversial pathogenesis. Although metronidazole (Mz) is standard therapy for Blastocystis infections, there have been accumulating reports of treatment failure, suggesting the existence of drug-resistant isolates. Furthermore, very little is known about Blastocystis susceptibility to standard antimicrobials. In the present study, we established resazurin and XTT viability microassays for Blastocystis spp. belonging to subtypes 4 and 7, both of which have been suggested to represent pathogenic zoonotic subtypes. The optimized resazurin assay was used to screen a total of 19 compounds against both subtypes. Interestingly, subtype 7 parasites were resistant to Mz, a 1-position-substituted 5-nitroimidazole (5-NI), while subtype 4 parasites were sensitive. Some cross-resistance was observed to tinidazole, another 1-position 5-NI. Conversely, subtype 4 parasites were resistant to emetine, while subtype 7 parasites were sensitive. Position 2 5-NIs were effective against both subtypes, as were ornidazole, nitazoxanide, furazolidone, mefloquine, quinicrine, quinine, cotrimoxazole (trimethoprim-sulfamethoxazole), and iodoacetamide. Both subtypes were resistant to chloroquine, doxycycline, paromomycin, ampicillin, and pyrimethamine. This is the first study to report extensive variations in drug sensitivities among two clinically important subtypes. Our study highlights the need to reevaluate established treatment regimens for Blastocystis infections and offers clear new treatment options for Mz treatment failures.

The roles of parasite-derived extracellular vesicles in disease and host-parasite communication.

In recent years, several parasites have been shown to interact with their hosts through intra- and inter-community communication mechanisms, which were identified to be mediated by extracellular vesicles (EVs) through various uptake mechanisms. EVs are a heterogenous group of nanoparticles (~30-5000 nm) classified into three main types according to their size and biogenesis. EVs contain proteins, lipids, nucleic acids and metabolites from the cell of origin which are essential for genetic exchange, biomarker identification and diagnosis of pathological diseases. As important "forward lines of parasite infectivity", the parasite-secreted EVs function as information transmitters in the early-stage of host-parasite interaction and subsequent host-cell colonization. For this review, we summarize from the literature the relevance of EVs to the pathogenesis and development of human parasitic protistan diseases such as giardiasis, leishmaniasis, amoebiasis, malaria and Blastocystis-mediated gut pathology. Specific in vitro and in vivo interactions of the parasite-EVs and the host, with the reported cellular and immunological outcomes are discussed in this review. EVs have great potential to be further developed as diagnostic, immunomodulation and therapeutic alternatives to fill the knowledge gaps in the current parasitic diseases discussed in this review. Nanomedicine and vaccine development could be explored, with the utilization and/or modification of the parasitic EVs as novel treatment and prevention strategies.

Combination Treatment With Remdesivir and Ivermectin Exerts Highly Synergistic and Potent Antiviral Activity Against Murine Coronavirus Infection.

The recent COVID-19 pandemic has highlighted the urgency to develop effective antiviral therapies against the disease. Murine hepatitis virus (MHV) is a coronavirus that infects mice and shares some sequence identity to SARS-CoV-2. Both viruses belong to the Betacoronavirus genus, and MHV thus serves as a useful and safe surrogate model for SARS-CoV-2 infections. Clinical trials have indicated that remdesivir is a potentially promising antiviral drug against COVID-19. Using an in vitro model of MHV infection of RAW264.7 macrophages, the safety and efficacy of monotherapy of remdesivir, chloroquine, ivermectin, and doxycycline were investigated. Of the four drugs tested, remdesivir monotherapy exerted the strongest inhibition of live virus and viral RNA replication of about 2-log10 and 1-log10, respectively (at 6 µM). Ivermectin treatment showed the highest selectivity index. Combination drug therapy was also evaluated using remdesivir (6 µM) together with chloroquine (15 µM), ivermectin (2 µM) or doxycycline (15 µM) - above their IC50 values and at high macrophage cell viability of over 95%. The combination of remdesivir and ivermectin exhibited highly potent synergism by achieving significant reductions of about 7-log10 of live virus and 2.5-log10 of viral RNA in infected macrophages. This combination also resulted in the lowest cytokine levels of IL-6, TNF-α, and leukemia inhibitory factor. The next best synergistic combination was remdesivir with doxycycline, which decreased levels of live virus by ~3-log10 and viral RNA by ~1.5-log10. These results warrant further studies to explore the mechanisms of action of the combination therapy, as well as future in vivo experiments and clinical trials for the treatment of SARS-CoV-2 infection.

Prevalence and molecular subtyping of Blastocystis in patients with Clostridium difficile infection, Singapore.

BACKGROUND: Blastocystis is a common anaerobic colonic protist in humans with controversial pathogenicity. Clostridium difficile (C. difficile) is the commonest cause of infectious diarrhea in healthcare settings. The prevalence and subtype (ST) characteristics of Blastocystis in patients with C. difficile infection (CDI) are rarely documented. Therefore, the present study was conducted to investigate the prevalence and subtype characteristics of Blastocystis in patients with suspicion of CDI in Singapore. METHODS: Fecal samples were collected from 248 patients presenting with suspected CDI from a single tertiary hospital in Singapore. C. difficile was diagnosed through positive glutamate dehydrogenase (GDH) with or without toxin A/B using enzyme immunoassay methods. The prevalence and subtype genetic characteristics of Blastocystis were determined by polymerase chain reaction (PCR) amplification and analysis of the barcode region of the SSU rRNA gene. RESULTS: The proportion of C. difficile in patients with healthcare-associated diarrhea in this study was 44% (109/248). Among the 109 C. difficile-positive patients, 59 (54.1%, 59/109) tested positive for toxigenic C. difficile, which was considered CDI. Based on the sequence analyses of the barcode region of the SSU rRNA gene, 10.1% (25/248) of the patients were found to be Blastocystis-positive, and three subtypes were identified: ST7 (64%, 16/25), ST1 (20%, 5/25), and ST3 (16%, 4/25). Remarkably, we found five patients with Blastocystis and C. difficile coinfection, and further subtype analysis showed two with ST7, two with ST1, and one with ST3. CONCLUSIONS: To the best of our knowledge, this is the first study to investigate the subtype distributions of Blastocystis in patients with CDI in Singapore. We found ST7 to be the predominant subtype in diarrheal patients. The pathogenicity of ST7 has been strongly suggested in previous in vitro and mouse model experiments, further confirming its potential pathogenicity to humans.

High-Content Phenotypic Screen of a Focused TCAMS Drug Library Identifies Novel Disruptors of the Malaria Parasite Calcium Dynamics.

The search for new antimalarial drugs with unexplored mechanisms of action is currently one of the main objectives to combat the resistance already in the clinic. New drugs should target specific mechanisms that once initiated lead inevitably to the parasite's death and clearance and cause minimal toxicity to the host. One such new mode of action recently characterized is to target the parasite's calcium dynamics. Disruption of the calcium homeostasis is associated with compromised digestive vacuole membrane integrity and release of its contents, leading to programmed cell death-like features characterized by loss of mitochondrial membrane potential and DNA degradation. Intriguingly, chloroquine (CQ)-treated parasites were previously reported to exhibit such cellular features. Using a high-throughput phenotypic screen, we identified 158 physiological disruptors (hits) of parasite calcium distribution from a small subset of approximately 3000 compounds selected from the GSK TCAMS (Tres Cantos Anti-Malarial Set) compound library. These compounds were then extensively profiled for biological activity against various CQ- and artemisinin-resistant Plasmodium falciparum strains and stages. The hits were also examined for cytotoxicity, speed of antimalarial activity, and their possible inhibitory effects on heme crystallization. Overall, we identified three compounds, TCMDC-136230, -125431, and -125457, which were potent in inducing calcium redistribution but minimally inhibited heme crystallization. Molecular superimposition of the molecules by computational methods identified a common pharmacophore, with the best fit assigned to TCMDC-125457. There were low cytotoxicity or CQ cross-resistance issues for these three compounds. IC50 values of these three compounds were in the low micromolar range. In addition, TCMDC-125457 demonstrated high efficacy when pulsed in a single-dose combination with artesunate against tightly synchronized artemisinin-resistant ring-stage parasites. These results should add new drug options to the current armament of antimalarial drugs as well as provide promising starting points for development of drugs with non-classical modes of action.