Pathogenic Protozoan Parasites Research Articles

Heterologous expression and biochemical characterization of the recombinant nucleoside triphosphate diphosphohydrolase 2 (LbNTPDase2) from Leishmania (Viannia) braziliensis.

Leishmania braziliensis is a pathogenic protozoan parasite that causes American Tegumentary Leishmaniasis (ATL), an important tropical neglected disease. ENTPDases are nucleotidases that hydrolyze intracellular and/or extracellular nucleotides. ENTPDases are known as regulators of purinergic signalling induced by extracellular nucleotides. Leishmania species have two isoforms of ENTPDase, and, particularly, ENTPDase2 seems to be involved in infectivity and virulence. In this study, we conducted the heterologous expression and biochemical characterization of the recombinant ENTPDase2 of L. braziliensis (rLbNTPDase2). Our results show that this enzyme is a canonical ENTPDase with apyrase activity, capable of hydrolysing triphosphate and diphosphate nucleotides, and it is dependent on divalent cations (calcium or magnesium). Substrate specificity was characterized as UDP>GDP>ADP>GTP>ATP=UTP. The enzyme showed optimal activity at a neutral to basic pH and was partially inhibited by suramin and DIDS. Furthermore, the low apparent Km for ADP suggests that the enzyme may play a role in adenosine-mediated signalling. The biochemical characterization of this enzyme can open new avenues for using LbNTPDase2 as a drug target.

A high-dimensional platform for observing neutrophil-parasite interactions.

Diarrheal diseases with infectious etiology remain a major cause of death globally, particularly in low-income countries. Entamoeba histolytica is a pathogenic protozoan parasite that is the causative agent of amebiasis. Amebiasis has a wide presentation in clinical severity with many factors, including the bacterial microbiota, contributing to this variation. The innate immune response also plays a critical role in regulating the severity of E. histolytica infection, with neutrophils reported to have a protective role. Despite this, the precise mechanism of how neutrophils mediate amebic killing is poorly understood. Thus, modern platforms that allow for inquiry of granulocyte-ameba interactions will increase our understanding of this disease. Herein, we describe an assay for neutrophil killing of E. histolytica by utilizing high-dimensional spectral flow cytometry. Neutrophils were isolated from wild-type 5-week-old C57BL/6 mice and co-cultured with E. histolytica at various multiplicity of infections (MOIs). After co-culture, neutrophils and E. histolytica were stained for spectral flow cytometry. Cell populations were identified using surface markers and fluorescence minus one (FMO) controls. We have previously shown that animals colonized with a component of the human microbiota, Clostridium scindens, were protected from E. histolytica. This protection was associated with elevated neutrophil count. Here, we explored amebic killing capacity and observed that neutrophils from animals with C. scindens possessed heightened amebic killing compared with controls. Thus, this study establishes a novel platform that can provide an in-depth analysis of granulocyte-parasite interactions in various contexts, including during alteration of the intestinal microbiota.IMPORTANCEThe tools for studying host immune cell-E. histolytica interactions are limited. Factors, such as parasite heterogeneity, infectivity, and difficulties with culture systems and animal models, make interrogation of these interactions challenging. Thus, Entamoeba researchers can benefit from next-generation models that allow for the analysis of both host and parasite cells. Here, we demonstrate the use of a novel platform that allows for the determination of parasite-host cell interactions and customizable high-dimensional phenotyping of both populations. Indeed, spectral flow cytometry can approach >40 markers on a single panel and can be paired with custom-developed parasite antibodies that can be conjugated to fluorochromes via commercially available kits. This platform affords researchers the capability to test highly precise hypotheses regarding host-parasite interactions.

Discovery of Strong 3-Nitro-2-Phenyl-2H-Chromene Analogues as Antitrypanosomal Agents and Inhibitors of Trypanosoma cruzi Glucokinase.

Chagas disease is one of the world's neglected tropical diseases, caused by the human pathogenic protozoan parasite Trypanosoma cruzi. There is currently a lack of effective and tolerable clinically available therapeutics to treat this life-threatening illness and the discovery of modern alternative options is an urgent matter. T. cruzi glucokinase (TcGlcK) is a potential drug target because its product, d-glucose-6-phosphate, serves as a key metabolite in the pentose phosphate pathway, glycolysis, and gluconeogenesis. In 2019, we identified a novel cluster of TcGlcK inhibitors that also exhibited anti-T. cruzi efficacy called the 3-nitro-2-phenyl-2H-chromene analogues. This was achieved by performing a target-based high-throughput screening (HTS) campaign of 13,040 compounds. The selection criteria were based on first determining which compounds strongly inhibited TcGlcK in a primary screen, followed by establishing on-target confirmed hits from a confirmatory assay. Compounds that exhibited notable in vitro trypanocidal activity over the T. cruzi infective form (trypomastigotes and intracellular amastigotes) co-cultured in NIH-3T3 mammalian host cells, as well as having revealed low NIH-3T3 cytotoxicity, were further considered. Compounds GLK2-003 and GLK2-004 were determined to inhibit TcGlcK quite well with IC50 values of 6.1 µM and 4.8 µM, respectively. Illuminated by these findings, we herein screened a small compound library consisting of thirteen commercially available 3-nitro-2-phenyl-2H-chromene analogues, two of which were GLK2-003 and GLK2-004 (compounds 1 and 9, respectively). Twelve of these compounds had a one-point change from the chemical structure of GLK2-003. The analogues were run through a similar primary screening and confirmatory assay protocol to our previous HTS campaign. Subsequently, three in vitro biological assays were performed where compounds were screened against (a) T. cruzi (Tulahuen strain) infective form co-cultured within NIH-3T3 cells, (b) T. brucei brucei (427 strain) bloodstream form, and (c) NIH-3T3 host cells alone. We report on the TcGlcK inhibitor constant determinations, mode of enzyme inhibition, in vitro antitrypanosomal IC50 determinations, and an assessment of structure-activity relationships. Our results reveal that the 3-nitro-2-phenyl-2H-chromene scaffold holds promise and can be further optimized for both Chagas disease and human African trypanosomiasis early-stage drug discovery research.

Prevalence And Associated Risk Factors With Entamoeba histolytica Infections In School Children

Entamoeba histolytica is medically important pathogenic parasitic protozoan of human in origin which causes amoebiasis that is common in children of devolving nations in tropical and sub-tropical regions of the world. A total of 100 children, comprising 45 boys and 55 girls, were screened to calculate the prevalence and risk factors of E.histolytica along with their socio-demographic characteristics among school children at Chkadara, Lower Dir, Pakistan. The stool samples were collected and transported to the Parasitology Laboratory, Zoology Separtment, Malakand University for parasitic investigation. Each of the stool samples were processed through floatation and sedimentation procedures and techniques and examined under the microscope first under low and then high power objectives. The protozoan cysts and the helminth eggs were have been investigated and identified through valid keys. The data was analysed through chi-square test via GraphPad version 5. The p vale was considered statistically significant if less than 0.05 at 95% CI. The results indicates that Entamoeba histolytica is a prominent pathogenic protozoan parasite in school children, with a total of 67 cases identified. Boys showed a slightly higher prevalence of this parasite, with 58.8% (n=30/51) cases compared to 58.7% (n=37/63) in boys. Ascaris lumbricoides, is another common intestinal parasite, was observed in a total of 35 cases, (30.7%) with an almost equal distribution among boys (17 cases) and girls (18 cases). Moreover, hookworm species was found in 11 children, with a somewhat higher prevalence in girls 11.1% (7 cases). Giardia spp, although less prevalent, still accounted for 4 cases, equally distributed among boys. E.histolytica was highest in prevalence as 58.7 %( 67/114) followed by Ascaris lumbricoides 30.7 % (n=35/114), then hookworms 9.64 % (n=11/114) while Giardia spp was found in least prevalence 0.87 %( n=1/114). Pattern of infection reveal that most of the students were found to be infected with 2 species of parasites including one is E.histolytica (37%) followed by single infection 20% with E.histolytica while least number of participants were reported to be infected with 3 species of parasites 10%. The overall prevalence was reported as 67%. Age and types of stool were not significantly associated with the prevalence of E.histolytica P>0.05, however all other factors sex, diarrheal status, parent education, parent employment status and economic conditions were significantly related to the prevalence of E.histolytica infection (P<0.05).

Prevalence and assemblage of Giardia duodenalis in a case-control study of children under 5 years from Jimma, Southwest Ethiopia

Giardia duodenalis is a common pathogenic intestinal protozoan parasite with high prevalence in developing countries, especially among children. The distribution of giardia assemblages among humans and their clinical relevance remains controversial. This study aimed to determine the prevalence and assemblage of Giardia among children under 5 years of age in Jimma, Southwest Ethiopia. Employing a case-control design, 606 children presenting with diarrhea at Jimma university medical center and Serbo Health Center were enrolled from December 2016 to July 2018 along with 617 matched controls without diarrhea. Giardia was detected and typed using real-time PCR. Univariate and multivariate regression analysis was performed. The total prevalence of Giardia was 41% (501/1223) and did not differ significantly between cases and controls (40% vs 42%). Prevalence increased by age, with the highest prevalence seen in children aged ≥ 25 months. Children without diarrhea with a history of diarrhea during the last month were more likely to be Giardia positive compared to children with no history diarrhea (OR 1.8 and 95%CI; 1.1–2.9). Regardless of current diarrhea symptoms, assemblage B predominated with 89%, followed by assemblage A (8%) and mixed infection assemblage A and B (3%). We report a high prevalence of Giardia by PCR detection in Jimma, Ethiopia, with assemblage B being predominant. There was a similar distribution of Giardia assemblages between children with and without diarrhea. Increasing age was a risk factor for Giardia infection. Community-based prevention and control strategies need to be employed to decrease the risk of giardia infection.

Proteomic Analysis of Fractionated Eimeria tenella Sporulated Oocysts Reveals Involvement in Oocyst Wall Formation.

Eimeria tenella is the most pathogenic intracellular protozoan parasite of the Eimeria species. Eimeria oocyst wall biogenesis appears to play a central role in oocyst transmission. Proteome profiling offers insights into the mechanisms governing the molecular basis of oocyst wall formation and identifies targets for blocking parasite transmission. Tandem mass tags (TMT)-labeled quantitative proteomics was used to analyze the oocyst wall and sporocysts of E. tenella. A combined total of 2865 E. tenella proteins were identified in the oocyst wall and sporocyst fractions; among these, 401 DEPs were identified, of which 211 were upregulated and 190 were downregulated. The 211 up-regulated DEPs were involved in various biological processes, including DNA replication, fatty acid metabolism and biosynthesis, glutathione metabolism, and propanoate metabolism. Among these proteins, several are of interest for their likely role in oocyst wall formation, including two tyrosine-rich gametocyte proteins (EtGAM56, EtSWP1) and two cysteine-rich proteins (EtOWP2, EtOWP6). Concurrently, 96 uncharacterized proteins may also participate in oocyst wall formation. The present study significantly expands our knowledge of the proteome of the oocyst wall of E. tenella, thereby providing a theoretical basis for further understanding of the biosynthesis and resilience of the E. tenella oocyst wall.

Fenbendazole resistance in Heterakis gallinarum, the vector of Histomonas meleagridis, on a broiler breeder farm in South Carolina.

Parasites are highly prevalent in poultry; thus, the management of parasites is a key component in the profitable production of poultry. The most common nematode parasite of poultry, Heterakis gallinarum, typically causes no direct pathology but is the vector of Histomonas meleagridis, a highly pathogenic protozoan parasite that causes blackhead disease. There are no approved treatments for H. meleagridis, making control reliant on controlling the helminth vector. In the United States, the benzimidazole anthelmintic fenbendazole (FBZ) is the only approved treatment for H. gallinarum. We were contacted by an industry veterinarian regarding clinical problems with histomoniasis despite frequent anthelmintic treatments. Given that we had recently diagnosed FBZ resistance in the closely related parasite Ascaridia dissimilis, we were interested to determine if H. gallinarum had also evolved resistance. An initial on-farm pilot study using 20 birds suggested that FBZ was poorly effective, therefore a larger controlled study was initiated. Heterakis gallinarum eggs were isolated from litter at the farm and used to infect 118 chicks. Treatment groups included a non-treated control, a label-, and a 2×-label dose of FBZ, with 36 birds per group divided into two replicates of 18 birds. Three weeks post-hatch, birds were infected with 150 embryonated eggs. Two weeks post-infection treated birds were administered either a label- or 2× label-dose of FBZ in water for five days (SafeGuard® Aquasol, 1mg/kg BW). To increase the likelihood that all birds consumed the full intended dose, the dosage was calculated using 1.25 times the average body weight. One-week post-treatment, birds were euthanized, and parasites enumerated. There were no significant differences in worm numbers recovered from any of the three groups (p-value=0.3426), indicating that both dosages of FBZ failed to provide the expected levels of efficacy. These data provide strong evidence that H. gallinarum has developed resistance to FBZ on this farm. Consequently, on this farm, or any farm with FBZ-resistant H. gallinarum, H. meleagridis will continue to cycle in an unrestricted manner despite administration of anthelmintic treatments. Given recent evidence of increasing problems with histomoniasis, and the fact that resistance was documented on the first farm we investigated, further investigations are needed to determine the prevalence of resistance in H. gallinarum on poultry farms. These data, when viewed together with our recent findings of FBZ resistance in A. dissimilis on multiple farms, suggest that drug resistance in ascarid nematodes may be an emerging problem in the US poultry industry.

Diagnosis and control of cryptosporidiosis in farm animals.

Cryptosporidium is a pathogenic protozoan parasite infecting the gastrointestinal epithelium of human and animal hosts. In farm animals, cryptosporidiosis causes significant economic losses including deaths in newborn animals, retarded growth, increased labor involved and high cost of drugs. The detection of Cryptosporidium oocysts in fecal samples is traditionally dependent on examination of stained slides by light microscope or by advanced microscopical tools such as: electron microscopy and phase contrast microscopy. Immunological diagnosis using either antibody or antigen detection could offer high sensitivity and specificity. Examples for these tests are Enzyme Linked Immunosorbent Assay (ELISA), Immunochromatographic tests, Immunochromatographic lateral flow (ICLF), Immunofluorescence assays (IFA) and Flow cytometry coupled with cell sorting. Molecular methods could differentiate species and genotypes of Cryptosporidium and help in studying the epidemiological features of this parasite with rapid, simple and sensitive procedures. Nanotechnology-based platforms could improve the sensitivity and specificity of other detection methods like: ELISA, ICLF, IFA and polymerase chain reaction. As the available prophylactic and therapeutic drugs or natural products treatments are insufficient and no approved vaccines are available, the best approach to control this parasite is by following firm hygienic measures. Many vaccine attempts were performed using hyperimmune colostrum, live or attenuated vaccines, recombinant and Deoxyribonucleic acid vaccines. Also, Clustered Regularly Interspaced Short Palindromic Repeats/Cas9 technology could help in Cryptosporidium genome editing to improve drug and vaccine discovery. Another approach that could be useful for assigning drug targets is metabolomics. Probiotics were also used successfully in the treatment of acute diarrhea and they proved a limiting effect on cryptosporidiosis in animal models. In addition, nanotherapy-based approaches could provide a good strategy for improving the potency of any type of drugs against Cryptosporidium and give good anti-cryptosporidial effects. In conclusion, accurate diagnosis using advanced techniques is the key to the control and prevention of cryptosporidiosis.

STUDIES ON FORMULATION AND EVALUATION OF LIPOSOME FOR CUTANEOUS LEISHMANIASIS

Aim of present study was to design liposome for allopurinol delivery against cutaneous leishmaniasis (CL). Leishmaniasis is disease caused by pathogenic protozoan parasites of the genus Leishmania. The disease is complex and untreated cases of leishmaniasis may result to chronic form such as mucosal leishmaniasis. There are several therapeutics available to treat CL, however, in recent time allopurinol has been used to tackle the chronic cases. The conventional formulations of allopurinol remain associated with several limitations both pharmacokinetic and pharmacodynamic. In this study, nanodesign liposome has been used to enhance pharmacokinetic and pharmacodynamic parameters. The liposomes were prepared by dried thin film hydration technique using a rotary evaporator with drug and carrier (soybean lecithin). The prepared liposomes formulations were evaluated for physical and chemical characteristics like average vesicle size, shape, zeta potential, % free drug, assay and stability studies. This developed liposomal drug delivery system was also evaluated for in-vitro drug release studies by pH 7.4 phosphate buffer using membrane diffusion method. The cumulative percentage of drug release was reported highest in the LF4 i.e. 98.08±1.54. Additionally, ex vivo studies have shown that allopurinol loaded into liposome has higher permeation (2.68µg/cm2 ) compared to allopurinol alone. These finding demonstrate liposome based allopurinol based drug delivery is effective compared to conventional formations.

Whole transcriptome analysis provides new insight on immune response mechanism of golden pompano (Trachinotus ovatus) to Amyloodinium ocellatum infestation

Ectoparasitic dinoflagellate Amyloodinium ocellatum (AO) is one of the most pathogenic protozoan parasites, causing amyloodiniosis in temperate and tropical marine fish. Golden pompano is susceptible to AO and increasing outbreaks of AO have posed a major threat to the economic sustainability of intensive aquaculture industry. In this study, natural parasite infection was simulated, and whole transcriptome analysis was performed with infected golden pompano gills to explore the host immune response to AO infestation. Overall, 4388 differentially expressed (DE) genes, 395 DE long non-coding RNAs (lncRNAs), 10 DE circular RNAs (circRNAs) and 96 DE microRNAs (miRNAs) were identified. DE genes were involved in pathways of ECM-receptor interaction and cytokine-cytokine receptor interaction, including many crucial extracellular matrix (ECM) components (integrin, laminin and collagen), cytokines (interleukin and lymphotoxin) and C-C motif chemokines. DE lncRNAs were significantly enriched in the regulation of immune cells, such as monocytes, neutrophils and lymphocytes. DE circRNAs were notably correlated with NOD-like receptor signaling pathway. DE miRNAs were prominently associated with immune-related pathways of cytokine-cytokine receptor interaction, intestinal immune network for IgA production, herpes simplex infection and TGF-beta signaling pathway. Furthermore, a core competing endogenous RNA (ceRNA) network was constructed, consisting of 28 DE mRNAs, 21 DE miRNAs, 8 DE circRNAs and 58 DE lncRNAs. The key roles of cytokine-cytokine receptor interaction, focal adhesion, MAPK signaling, NOD-like receptor signaling and mTOR signaling pathway were highlighted in the ceRNA network. In total, the present study uncovered the molecular changes of coding and non-coding RNAs involved in host immune response induced by AO infestation. The results could contribute significantly to a comprehensive understanding of host immune response to AO infestation and provide new clues for developing efficient amyloodiniosis control measures and germplasm improvement in artificial breeding of golden pompano.

Host cell proteins modulated upon Toxoplasma infection identified using proteomic approaches: a molecular rationale.

Toxoplasma gondii is a pathogenic protozoan parasite belonging to the apicomplexan phylum that infects the nucleated cells of warm-blooded hosts leading to an infectious disease known as toxoplasmosis. Apicomplexan parasites such as T. gondii can display different mechanisms to control or manipulate host cells signaling at different levels altering the host subcellular genome and proteome. Indeed, Toxoplasma is able to modulate host cell responses (especially immune responses) during infection to its advantage through both structural and functional changes in the proteome of different infected cells. Consequently, parasites can transform the invaded cells into a suitable environment for its own replication and the induction of infection. Proteomics as an applicable tool can identify such critical proteins involved in pathogen (Toxoplasma)-host cell interactions and consequently clarify the cellular mechanisms that facilitate the entry of pathogens into host cells, and their replication and transmission, as well as the central mechanisms of host defense against pathogens. Accordingly, the current paper reviews several proteins (identified using proteomic approaches) differentially expressed in the proteome of Toxoplasma-infected host cells (macrophages and human foreskin fibroblasts) and tissues (brain and liver) and highlights their plausible functions in the cellular biology of the infected cells. The identification of such modulated proteins and their related cell impact (cell responses/signaling) can provide further information regarding parasite pathogenesis and biology that might lead to a better understanding of therapeutic strategies and novel drug targets.

Mining the Proteome of Toxoplasma Parasites Seeking Vaccine and Diagnostic Candidates.

Simple SummaryThe One Health concept to toxoplasmosis highlights that the health of humans is closely related to the health of animals and our common environment. Toxoplasmosis outcomes might be severe and fatal in patients with immunodeficiency, diabetes, and pregnant women and infants. Consequently, the development of effective vaccine and diagnostic strategies is urgent for the elimination of this disease. Proteomics analysis has allowed the identification of key proteins that can be utilized in the development of novel disease diagnostics and vaccines. This work presents relevant proteins found in the proteome of the life cycle-specific stages of Toxoplasma parasites. In fact, it brings together the main functionality key proteins from Toxoplasma parasites coming from proteomic approaches that are most likely to be useful in improving the disease management, and critically proposes innovative directions to finally develop promising vaccines and diagnostics tools.Toxoplasma gondii is a pathogenic protozoan parasite that infects the nucleated cells of warm-blooded hosts leading to an infectious zoonotic disease known as toxoplasmosis. The infection outcomes might be severe and fatal in patients with immunodeficiency, diabetes, and pregnant women and infants. The One Health approach to toxoplasmosis highlights that the health of humans is closely related to the health of animals and our common environment. The presence of drug resistance and side effects, the further improvement of sensitivity and specificity of serodiagnostic tools and the potentiality of vaccine candidates to induce the host immune response are considered as justifiable reasons for the identification of novel targets for the better management of toxoplasmosis. Thus, the identification of new critical proteins in the proteome of Toxoplasma parasites can also be helpful in designing and test more effective drugs, vaccines, and diagnostic tools. Accordingly, in this study we present important proteins found in the proteome of the life cycle-specific stages of Toxoplasma parasites that are potential diagnostic or vaccine candidates. The current study might help to understand the complexity of these parasites and provide a possible source of strategies and biomolecules that can be further evaluated in the pathobiology of Toxoplasma parasites and for diagnostics and vaccine trials against this disease.

Phylogenetic analysis of Eimeria tenella isolated from the litter of different chicken farms in Mymensingh, Bangladesh.

ABSTRACTBackgroundEimeria tenella is the most pathogenic intracellular protozoan parasite of seven Eimeria species causing chicken coccidiosis around the world. This species is particularly responsible for caecal coccidiosis leading to serious morbidity–mortality and financial loss in poultry production.MethodsThe present study explored the genetic diversity of E. tenella. Litter slurry was collected from 18 broiler farms located in Mymensingh district, Bangladesh. Litter samples were processed for oocyst isolation–identification using parasitological techniques followed by genomic DNA extraction from sporulated oocysts. For molecular analysis, the i n t e r n a l t r a n s c r i b e d s p a c e r 1 gene of E. tenella was amplified using species‐specific primers and sequenced. After editing and alignment, 263 bp sequences were used for analysis.ResultsGenetic analysis showed seven distinct genotypes and detected six single nucleotide polymorphisms among the 18 E. tenella isolates. The nucleotide and genotype diversity were 0.00507 and 0.8235, respectively. A phylogenetic tree was constructed with 66 sequences (seven studied genotypes and 59 reference sequences from GenBank database). The neighbour‐joining tree represented that the studied E. tenella isolates were grouped with reference E. tenella isolates with strong nodal support (100%) and the nucleotide sequences of E. tenella, E. necatrix, E. acervulina, E. brunetti, E. maxima, E. mitis and E. praecox formed separate clusters without any geographical boundaries.ConclusionsThis is the first study on the genetic analysis of E. tenella from Mymensingh district, Bangladesh. These findings will provide baseline data on the species conformation and genetic variations of E. tenella. Further extensive investigation will be needed to reveal the population genetic structure of this parasite and thus will facilitate the planning of effective control strategies.

Novel association between blastocystosis and some hemogram parameters in Iraqi people with and without irritable bowel syndrome.

Many studies have suggested that Blastocystis parasites are pathogenic protozoan parasites and one study proposed that these parasites have contributed to anaemia in children. The primary objective of the present study was to made a comparison between healthy control subjects (group 1), Blastocystis-positive subjects (group 2), subjects with irritable bowel syndrome (IBS) who were Blastocystis-negative (group 3), and IBS patients who were Blastocystispositive (group 4) regarding the haematological indices. From each participant, blood has been collected and the complete blood count (CBC) has been measured. The current study also was designed to evaluate the correlation between blastocystosis and six selected hemogram parameters [monocyte-to-lymphocyte ratio (MLR), neutrophil-tolymphocyte ratio (NLR), platelet-to-lymphocyte ratio (PLR), red cell distribution width (RDW), platelet distribution width (PDW), and mean platelet volume (MPV)]. The results revealed that the total numbers of lymphocytes and platelets decreased significantly (P<0.0001), while the numbers of monocytes (P<0.0001) and neutrophils (P<0.0001) increased significantly in the bloods of IBS patients and non-IBS subjects who were infected with Blastocystis parasites in comparison with the healthy subjects in the control group. In addition, our results revealed for the first time that the levels of NLR, MLR, PLR, RDW, PDW and MPV were significantly higher (P=0.05-0.0001) in the bloods of IBS patients and non-IBS subjects who were infected with Blastocystis parasites than in their Blastocystis-negative counterparts. In conclusion, we believe that the levels of these hemogram parameters can be used as novel markers of blastocystosis. In addition, this study revealed that the infection with Blastocystis parasites had a significant impact on the haematological indices in both IBS patients and non-IBS individuals.

A hybrid sequencing and assembly strategy for generating culture free Giardia genomes.

Giardia duodenalis is a pathogenic intestinal protozoan parasite of humans and many other animals. Giardia duodenalis is found throughout the world, and infection is known to have adverse health consequences for human and other mammalian hosts. Yet, many aspects of the biology of this ubiquitous parasite remain unresolved. Whole genome sequencing and comparative genomics can provide insight into the biology of G. duodenalis by helping to reveal traits that are shared by all G. duodenalis assemblages or unique to an individual assemblage or strain. However, these types of analyses are currently hindered by the lack of available G. duodenalis genomes, due, in part, to the difficulty in obtaining the genetic material needed to perform whole genome sequencing. In this study, a novel approach using a multistep cleaning procedure coupled with a hybrid sequencing and assembly strategy was assessed for use in producing high quality G. duodenalis genomes directly from cysts obtained from feces of two naturally infected hosts, a cat and dog infected with assemblage A and D, respectively. Cysts were cleaned and concentrated using cesium chloride gradient centrifugation followed by immunomagnetic separation. Whole genome sequencing was performed using both Illumina MiSeq and Oxford Nanopore MinION platforms. A hybrid assembly strategy was found to produce higher quality genomes than assemblies from either platform alone. The hybrid G. duodenalis genomes obtained from fecal isolates (cysts) in this study compare favorably for quality and completeness against reference genomes of G. duodenalis from cultured isolates. The whole genome assembly for assemblage D is the most contiguous genome available for this assemblage and is an important reference genome for future comparative studies. The data presented here support a hybrid sequencing and assembly strategy as a suitable method to produce whole genome sequences from DNA obtained from G. duodenalis cysts which can be used to produce novel reference genomes necessary to perform comparative genomics studies of this parasite.

Exploration of Pyrazolo[1,5-a]pyrimidines as Membrane-Bound Pyrophosphatase Inhibitors.

Inhibition of membrane‐bound pyrophosphatase (mPPase) with small molecules offer a new approach in the fight against pathogenic protozoan parasites. mPPases are absent in humans, but essential for many protists as they couple pyrophosphate hydrolysis to the active transport of protons or sodium ions across acidocalcisomal membranes. So far, only few nonphosphorus inhibitors have been reported. Here, we explore the chemical space around previous hits using a combination of screening and synthetic medicinal chemistry, identifying compounds with low micromolar inhibitory activities in the Thermotoga maritima mPPase test system. We furthermore provide early structure‐activity relationships around a new scaffold having a pyrazolo[1,5‐a]pyrimidine core. The most promising pyrazolo[1,5‐a]pyrimidine congener was further investigated and found to inhibit Plasmodium falciparum mPPase in membranes as well as the growth of P. falciparum in an ex vivo survival assay.

The IL-33-ILC2 pathway protects from amebic colitis

Entamoeba histolytica is a pathogenic protozoan parasite that causes intestinal colitis, diarrhea, and in some cases, liver abscess. Through transcriptomics analysis, we observed that E. histolytica infection was associated with increased expression of IL-33 mRNA in both the human and murine colon. IL-33, the IL-1 family cytokine, is released after cell injury to alert the immune system of tissue damage. Treatment with recombinant IL-33 protected mice from amebic infection and intestinal tissue damage; moreover, blocking IL-33 signaling made mice more susceptible to amebiasis. IL-33 limited the recruitment of inflammatory immune cells and decreased the pro-inflammatory cytokine IL-6 in the cecum. Type 2 immune responses were upregulated by IL-33 treatment during amebic infection. Interestingly, administration of IL-33 protected RAG2–/– mice but not RAG2−/−γc−/− mice, demonstrating that IL-33-mediated protection required the presence of innate lymphoid cells (ILCs). IL-33 induced recruitment of ILC2 but not ILC1 and ILC3 in RAG2−/− mice. At baseline and after amebic infection, there was a significantly higher IL13+ILC2s in C57BL/J mice, which are naturally resistant to amebiasis, than CBA/J mice. Adoptive transfer of ILC2s to RAG2−/−γc−/− mice restored IL-33-mediated protection. These data reveal that the IL-33-ILC2 pathway is an important host defense mechanism against amebic colitis.

Leishmaniosis in Rodents Caused by Leishmania infantum: A Review of Studies in the Mediterranean Area.

Leishmaniosis infection begins when a phlebotomine sand fly vector inoculates pathogenic protozoan parasites of the genus Leishmania into a mammalian host. In the case of Leishmania infantum, the domestic dog is considered to be the main parasite reservoir, and canine leishmaniosis (CanL) has a high mortality rate in untreated dogs. Hundreds of cases of human leishmaniosis (HL) are reported in the world each year, the incidence in Europe being relatively low. Leishmaniosis control is primarily focused on the dog, combining methods that prevent sand fly bites and boost host resistance to infection. However, these measures are only partially effective and new solutions need to be found. One of the main factors limiting CanL and HL control is the existence of a sylvatic Leishmania transmission cycle that interacts with the domestic cycle maintained by dogs. It is suspected that the main reservoir of infection in wildlife are rodents, whose expansion and rapid population growth worldwide is increasing the risk of human and zoonotic pathogen transfer. The aim of this review is therefore to analyze reports in the literature that may shed light on the potential role of rodents in the leishmaniosis transmission cycle in the Mediterranean area. Following the general methodology recommended for reviews, six databases (Google Scholar, Ovid, PubMed, Science Direct, Scopus and Web of Science) were explored for the period January 1995 to December 2020. The results extracted from 39 publications that met the established inclusion criteria were analyzed. It was found that 23 species of rodents have been studied in nine countries of the Mediterranean basin. Of the 3,643 specimens studied, 302 tested positive for L. infantum infection by serology, microscopy and/or molecular techniques.

Entamoeba histolytica Adaption to Auranofin: A Phenotypic and Multi-Omics Characterization.

Auranofin (AF), an antirheumatic agent, targets mammalian thioredoxin reductase (TrxR), an important enzyme controlling redox homeostasis. AF is also highly effective against a diversity of pathogenic bacteria and protozoan parasites. Here, we report on the resistance of the parasite Entamoeba histolytica to 2 µM of AF that was acquired by gradual exposure of the parasite to an increasing amount of the drug. AF-adapted E. histolytica trophozoites (AFAT) have impaired growth and cytopathic activity, and are more sensitive to oxidative stress (OS), nitrosative stress (NS), and metronidazole (MNZ) than wild type (WT) trophozoites. Integrated transcriptomics and redoxomics analyses showed that many upregulated genes in AFAT, including genes encoding for dehydrogenase and cytoskeletal proteins, have their product oxidized in wild type trophozoites exposed to AF (acute AF trophozoites) but not in AFAT. We also showed that the level of reactive oxygen species (ROS) and oxidized proteins (OXs) in AFAT is lower than that in acute AF trophozoites. Overexpression of E. histolytica TrxR (EhTrxR) did not protect the parasite against AF, which suggests that EhTrxR is not central to the mechanism of adaptation to AF.

Comparison of Different Methods of Detection of Enteric Pathogenic Protozoa

Purpose: The study was conducted to compare different methods of detection of pathogenic protozoan parasites in stool specimens of People Living with HIV/AIDS (PLHA). Materials and Methods: Stool specimens of 242 HIV sero-positive patients were examined using the wet mount technique, modified Ziehl-Neelsen’s (ZN) staining, auto-fluorescence and auramine fluorescence staining. Patient specimens, 94 and 40 out of 242, were also subjected to Giardia antigen detection using an enzyme immunoassay and Cryptosporidium antigen detection by immuno-chromatography, respectively. For calculation of sensitivity, specificity, positive and negative predictive values, light microscopy of wet mounts and modified ZN stained smears for Giardia and Coccidia, respectively, were considered as gold standards. Results: Sensitivity of auto-fluorescence, auramine-O staining and antigen detection techniques was found to be 100% as compared to the routine standards. The specificity of auto-fluorescence was 90.6% and 100% for Cyclospora and Isospora, respectively; that of auramine-O staining was 98.9% for Cryptosporidium, 99.30% for Cyclospora and 100% for Isospora; and that of antigen detection was 90.6% and 97.7% for Cryptosporidium and Giardia, respectively. Conclusion: In laboratories requiring screening of large number of stool specimens for detection of protozoan parasites, fluorescence microscopy and antigen detection can be useful techniques. Confirmation of positive results, however, needs to be done with the standard techniques.