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This article examines the long-term outcomes of pacemaker therapy in extracardiac Fontan patients, who undergo a specific type of heart surgery. The study found that a small percentage of Fontan patients required a pacemaker, and these patients had a lower survival rate compared to those without a pacemaker. The primary causes of heart transplantation or death in patients with pacemakers were ventricular failure and complications related to the Fontan procedure. The study suggests that extracardiac Fontan patients should be closely monitored for long-term complications. [Extracted from the article]

This article discusses the long-term outcomes of the extracardiac Fontan procedure, which is typically performed in patients aged 2-5 years with univentricular heart conditions. However, in some cases, the procedure is delayed until adolescence or adulthood. The study examines eight patients who underwent the procedure at the age of 16 or older. The results show that the extracardiac Fontan procedure can effectively improve the quality of life in these patients. The study also highlights the safety of the procedure and the impressive improvement in the patients' overall well-being. [Extracted from the article]

This article examines the applicability of prognostic scores for decision-making in patients with critical left ventricular outflow tract (LVOT) obstruction and borderline left ventricle (LV) who undergo the Ross-Konno procedure. The study evaluated four scores (Rhodes, Discriminant, CHSS1, and CHSS2) in a cohort of 51 patients treated at a single center. The results showed significant differences between patients who underwent biventricular repair and those who underwent univentricular repair. The study concludes that the validity of these scores is limited in Ross-Konno patients, and using the proposed changes after the procedure may result in an overemphasis on biventricular repair and a loss of specificity. [Extracted from the article]

The assembly and function of side chain modifications of glycosylphosphatidylinositol (GPI) units (anchors or free forms) are poorly defined. In a recent study, two enzymes, PIGJ and PIGE, of the protozoan parasite Toxoplasma gondii were identified and shown to be involved in the assembly of such GPI side chains (J. A. Alvarez, E. Gas-Pascual, S. Malhi, J. C. Sánchez-Arcila, et al., mBio 15:e00527-24, 2024, https://doi.org/10.1128/mbio.00527-24). PIGJ adds N-acetylgalactosamine to the GPI core structure, while PIGE subsequently adds a terminal glucose. Deletion of PIGJ resulted in the loss of the side chain and, strikingly, increased mortality in infected mice, in contrast to PIGE knockouts. Absence of the side chain led to increased binding of the scavenger receptor CD36 to mutant parasites. In galectin-3 knockout mice, the virulent phenotype of side-chain-deficient parasites was largely lost. While the exact mechanisms remain to be elucidated by more experiments, these findings provide the first evidence for the importance of GPI side chains in parasite-host interactions in vivo ., Competing Interests: The author declares no conflict of interest.

In a representative sample of female children and adolescents in Germany, Toxoplasma gondii seroprevalence was 6.3% (95% CI 4.7%-8.0%). With each year of life, the chance of being seropositive increased by 1.2, indicating a strong force of infection. Social status and municipality size were found to be associated with seropositivity.

Objective: Surgery for recurrent aortic arch obstruction is highly challenging and publications are rare. The aim of this retrospective, single-center study was to evaluate mortality, complications, and reintervention rate after an anatomic repair., Methods: Between 1999 and 2022, in total 946 operations on the aortic arch were performed at the Children's Heart Center Linz. In 39 cases, the indication was a recurrent or residual aortic arch obstruction or coarctation in a patient aged 18 years or younger. This is our study cohort. The aorta was reconstructed by a direct anastomosis/autograft in 20 patients, patch in 17 patients, and interposition graft in 2 adolescents. In 32 procedures, cardiopulmonary bypass with whole body perfusion was employed, in 4, antegrade cerebral perfusion was employed, in 2, a left heart bypass was employed, and in 1 no cardiopulmonary bypass was used., Results: Median (Q1, Q3) age at operation was 253 days (100, 2198 days), weight 7.5 kg (4.5, 17.8 kg). Median cardiopulmonary bypass time was 177 minutes (115, 219 minutes), crossclamp time 73 minutes (49, 102 minutes). Three infants died during the hospital stay: 1 with Williams syndrome, 1 with hypoplastic left heart syndrome, and 1 with heterotaxia. There was no death due to an arch complication. The main complications were 1 neurologic injury after postoperative resuscitation (Williams syndrome) and 1 permanent recurrent laryngeal nerve paralysis. During the follow-up period of median 8.1 years (2.6, 12 years) 1 re-reintervention on the aortic arch was necessary., Conclusions: Sophisticated reoperations on the aortic arch could be performed safely. In children, the growth potential of all segments of the aorta could be sustainably preserved by avoiding interposition or extra-anatomic bypass grafts., Competing Interests: The authors reported no conflicts of interest. The Journal policy requires editors and reviewers to disclose conflicts of interest and to decline handling manuscripts for which they may have a conflict of interest. The editors and reviewers of this article have no conflicts of interest., (© 2024 The Author(s).)

Objectives: Patients after the Norwood procedure are prone to postoperative instability. Extracorporeal membrane oxygenation (ECMO) can help to overcome short-term organ failure. This retrospective single-centre study examines ECMO weaning, hospital discharge and long-term survival after ECMO therapy between Norwood and bidirectional Glenn palliation as well as risk factors for mortality., Methods: In our institution, over 450 Norwood procedures have been performed. Since the introduction of ECMO therapy, 306 Norwood operations took place between 2007 and 2022, involving ECMO in 59 cases before bidirectional Glenn. In 48.3% of cases, ECMO was initiated intraoperatively post-Norwood. Patient outcomes were tracked and mortality risk factors were analysed using uni- and multivariable testing., Results: ECMO therapy after Norwood (median duration: 5 days; range 0-17 days) saw 31.0% installed under CPR. Weaning was achieved in 46 children (78.0%), with 55.9% discharged home after a median of 45 (36-66) days. Late death occurred in 3 patients after 27, 234 and 1541 days. Currently, 30 children are in a median 4.8 year (3.4-7.7) follow-up. At the time of inquiry, 1 patient awaits bidirectional Glenn, 6 are at stage II palliation, Fontan was completed in 22 and 1 was lost to follow-up post-Norwood. Risk factor analysis revealed dialysis (P < 0.001), cerebral lesions (P = 0.026), longer ECMO duration (P = 0.002), cardiac indication and lower body weight (P = 0.038) as mortality-increasing factors. The 10-year mortality probability after ECMO therapy was 48.5% (95% CI 36.5-62.9%)., Conclusions: ECMO therapy in critically ill patients after the Norwood operation may significantly improve survival of a patient cohort otherwise forfeited and give the opportunity for successful future-stage operations., (© The Author(s) 2024. Published by Oxford University Press on behalf of the European Association for Cardio-Thoracic Surgery. All rights reserved.)

Toxoplasma gondii is a zoonotic protozoan parasite and one of the most successful foodborne pathogens. Upon infection and dissemination, the parasites convert into the persisting, chronic form called bradyzoites, which reside within cysts in muscle and brain tissue. Despite their importance, bradyzoites remain difficult to investigate directly, owing to limited in vitro models. In addition, the need for new drugs targeting the chronic stage, which is underlined by the lack of eradicating treatment options, remains difficult to address since in vitro access to drug-tolerant bradyzoites remains limited. We recently published the use of a human myotube-based bradyzoite cell culture system and demonstrated its applicability to investigate the biology of T. gondii bradyzoites. Encysted parasites can be functionally matured during long-term cultivation in these immortalized cells and possess many in vivo-like features, including pepsin resistance, oral infectivity, and antifolate resistance. In addition, the system is scalable, enabling experimental approaches that rely on large numbers, such as metabolomics. In short, we detail the cultivation of terminally differentiated human myotubes and their subsequent infection with tachyzoites, which then mature to encysted bradyzoites within four weeks at ambient CO2 levels. We also discuss critical aspects of the procedure and suggest improvements. Key features • This protocol describes a scalable human myotube-based in vitro system capable of generating encysted bradyzoites featuring in vivo hallmarks. • Bradyzoite differentiation is facilitated through CO 2 depletion but without additional artificial stress factors like alkaline pH. • Functional maturation occurs over four weeks., Competing Interests: Competing interestsThe authors declare no competing interests., (©Copyright : © 2024 The Authors; This is an open access article under the CC BY license.)

Toxoplasmosis is one of the major foodborne parasitic diseases in Germany, with 49% of its population chronically infected with its causative agent, Toxoplasma gondii. Although the acute disease is usually benign in immunocompetent individuals, it is a threat for immunocompromised patients as well as for fetuses of seronegative mothers. As a result of infection, congenital and ocular toxoplasmosis can have serious lifelong consequences. Here I will highlight the epidemiologic situation, from its past in the two separate parts of Germany, to its unification 30 years ago and up to the present day. The main identified risk factor for infection in Germany is thought to be the consumption of undercooked or raw meat or sausages. However, the relative impact of this risky eating habit as well as that of other risk factors are changing and are discussed and compared to the situation in the Netherlands. Finally, the importance of robust and efficient high-throughput serological assays for obtaining reliable epidemiological data, on which public health decisions can be made, is highlighted. The potential of bead-based multiplex assays, which allow the incorporation of multiple antigens with different analytical properties and thus yield additional information, are described in this context. It illustrates the interdependence of new analytic assay developments and sound epidemiology, a foundation that decades-old data from Germany did not have., Competing Interests: Declarations of Competing Interest None., (Copyright © 2023 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Objectives: Stethoscopes carry a significant risk for pathogen transmission. Here, the safe use and performance of a new, non-sterile, single-use stethoscope cover (SC), that is impermeable for pathogens, was investigated by different healthcare professionals (HCPs) in the postoperative care setting of an intensive care unit (ICU)., Methods: Fifty-four patients underwent routine auscultations with the use of the SC (Stethoglove ® , Stethoglove GmbH, Hamburg, Germany). The participating HCPs ( n  = 34) rated each auscultation with the SC on a 5-point Likert scale. The mean ratings of acoustic quality and the SC handling were defined as primary and secondary performance endpoint., Results: 534 auscultations with the SC were performed (average 15.7/user) on the lungs (36.1%), the abdomen (33.2%), the heart (28.8%), or other body-sites (1.9%). No adverse device-effects occurred. The acoustic quality was rated at 4.2 ± 0.7 (mean) with a total of 86.1% of all auscultations being rated at least as 4/5, and with no rating as below 2. The SC handling was rated at 3.7 ± 0.8 (mean) with a total of 96.4% of all auscultations being rated at least 3/5., Conclusion: Using a real-world setting, this study demonstrates that the SC can be safely and effectively used as cover for stethoscopes during auscultation. The SC may therefore represent a useful and easy-to-implement tool for preventing stethoscope-mediated infections. Study Registration : EUDAMED no. CIV-21-09-037762., Competing Interests: ME is a scientific advisor to Stethoglove GmbH. Outside the submitted work: VF has relevant (institutional) financial activities with following commercial entities: Medtronic GmbH, Biotronik SE & Co., Abbott GmbH & Co. KG, Boston Scientific, Edwards Lifesciences, Berlin Heart, Novartis Pharma GmbH, JOTEC GmbH and Zurich Heart in relation to educational Grants (including travel support), fees for lectures and speeches, fees for professional consultation and research and study funds. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Nazari-Shafti, Meyborg, Iske, Schloss, Seeber, Friedrich, Exarchos, Richter, Falk and Emmert.)

Toxoplasma gondii oocysts, which are shed in large quantities in the feces from infected felines, are very stable in the environment, resistant to most inactivation procedures, and highly infectious. The oocyst wall provides an important physical barrier for sporozoites contained inside oocysts, protecting them from many chemical and physical stressors, including most inactivation procedures. Furthermore, sporozoites can withstand large temperature changes, even freeze-thawing, as well as desiccation, high salinity, and other environmental insults; however, the genetic basis for this environmental resistance is unknown. Here, we show that a cluster of four genes encoding Late Embryogenesis Abundant (LEA)-related proteins are required to provide Toxoplasma sporozoites resistance to environmental stresses. Toxoplasma LEA-like genes ( TgLEAs ) exhibit the characteristic features of intrinsically disordered proteins, explaining some of their properties. Our in vitro biochemical experiments using recombinant TgLEA proteins show that they have cryoprotective effects on the oocyst-resident lactate dehydrogenase enzyme and that induced expression in E. coli of two of them leads to better survival after cold stress. Oocysts from a strain in which the four LEA genes were knocked out en bloc were significantly more susceptible to high salinity, freezing, and desiccation compared to wild-type oocysts. We discuss the evolutionary acquisition of LEA -like genes in Toxoplasma and other oocyst-producing apicomplexan parasites of the Sarcocystidae family and discuss how this has likely contributed to the ability of sporozoites within oocysts to survive outside the host for extended periods. Collectively, our data provide a first molecular detailed view on a mechanism that contributes to the remarkable resilience of oocysts against environmental stresses. IMPORTANCE Toxoplasma gondii oocysts are highly infectious and may survive in the environment for years. Their resistance against disinfectants and irradiation has been attributed to the oocyst and sporocyst walls by acting as physical and permeability barriers. However, the genetic basis for their resistance against stressors like changes in temperature, salinity, or humidity, is unknown. We show that a cluster of four genes encoding Toxoplasma Late Embryogenesis Abundant (TgLEA)-related proteins are important for this resistance to environmental stresses. TgLEAs have features of intrinsically disordered proteins, explaining some of their properties. Recombinant TgLEA proteins show cryoprotective effects on the parasite's lactate dehydrogenase, an abundant enzyme in oocysts, and expression in E. coli of two TgLEAs has a beneficial effect on growth after cold stress. Moreover, oocysts from a strain lacking all four TgLEA genes were more susceptible to high salinity, freezing, and desiccation compared to wild-type oocysts, highlighting the importance of the four TgLEAs for oocyst resilience.

The full life cycle of Toxoplasma gondii cannot be recapitulated in vitro , and access to certain stages, such as mature tissue cysts (bradyzoites) and oocysts (sporozoites), traditionally requires animal experiments. This has greatly hindered the study of the biology of these morphologically and metabolically distinct stages, which are essential for the infection of humans and animals. However, several breakthrough advances have been made in recent years towards obtaining these life stages in vitro , such as the discovery of several molecular factors that induce differentiation and commitment to the sexual cycle, and different culture methods that use, for example, myotubes and intestinal organoids to obtain mature bradyzoites and different sexual stages of the parasite. We review these novel tools and approaches, highlight their limitations and challenges, and discuss what research questions can already be answered with these models. We finally identify future routes for recapitulating the entire sexual cycle in vitro ., Competing Interests: The authors have no personal or financial competing interests to declare.No competing interests were disclosed.No competing interests were disclosed.No competing interests were disclosed., (Copyright: © 2023 Seeber F et al.)

The apicoplast, a relict plastid found in most species of the phylum Apicomplexa, harbors the ferredoxin redox system which supplies electrons to enzymes of various metabolic pathways in this organelle. Recent reports in Toxoplasma gondii and Plasmodium falciparum have shown that the iron-sulfur cluster (FeS)-containing ferredoxin is essential in tachyzoite and blood-stage parasites, respectively. Here we review ferredoxin's crucial contribution to isoprenoid and lipoate biosynthesis as well as tRNA modification in the apicoplast, highlighting similarities and differences between the two species. We also discuss ferredoxin's potential role in the initial reductive steps required for FeS synthesis as well as recent evidence that offers an explanation for how NADPH required by the redox system might be generated in Plasmodium spp., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Objectives: A novel hybrid non-covered stent was developed to treat malperfusion and prevent aneurysm formation following hemiarch procedure for DeBakey I acute aortic dissection (AAD). The present analysis investigates the performance of the device in 100 consecutive implantations., Methods: Between 2018 and 2021, 100 patients underwent surgical repair of DeBakey I AAD with implantation of a non-covered stent in the arch and descending aorta. The primary entry tear was located in the root or the ascending aorta. Clinical and imaging data were collected and analysed retrospectively. The endpoints of the study were 30-day mortality, neurological outcome and need of additional procedures due to postoperative malperfusion. Technical success was assessed in the first postoperative computed tomography in regard to the induction of false lumen thrombosis in the descending aorta., Results: The median age was 61 (54-73) years. Preoperative malperfusion was present in 46 (46%) patients. The primary arterial cannulation strategy was the right axillary artery and an open distal anastomosis was performed in a median caudal circulatory arrest of 40 (34-52) min. In 48% of cases, a 55-40 tapered stent was implanted. The 30-day mortality was 18%, and the operation-related new postoperative neurological deficit was present in 8%. Technical success was achieved in 76% of patients., Conclusions: The novel non-covered stent can be safely applied to complement aortic repair with the hemiarch procedure for DeBakey I AAD. The expansion of the true lumen through the device may prevent postoperative malperfusion and induces positive vascular remodelling with the thrombosis of the false lumen., (© The Author(s) 2022. Published by Oxford University Press on behalf of the European Association for Cardio-Thoracic Surgery. All rights reserved.)

The apicomplexan parasite Toxoplasma gondii forms bradyzoite-containing tissue cysts that cause chronic and drug-tolerant infections. However, current in vitro models do not allow long-term culture of these cysts to maturity. Here, we developed a human myotube-based in vitro culture model of functionally mature tissue cysts that are orally infectious to mice and tolerate exposure to a range of antibiotics and temperature stresses. Metabolomic characterization of purified cysts reveals global changes that comprise increased levels of amino acids and decreased abundance of nucleobase- and tricarboxylic acid cycle-associated metabolites. In contrast to fast replicating tachyzoite forms of T. gondii these tissue cysts tolerate exposure to the aconitase inhibitor sodium fluoroacetate. Direct access to persistent stages of T. gondii under defined cell culture conditions will be essential for the dissection of functionally important host-parasite interactions and drug evasion mechanisms. It will also facilitate the identification of new strategies for therapeutic intervention., (© 2022. The Author(s).)

Objectives: The aim of this study was to externally validate a lab-based risk score (lactate, creatinine, aspartate aminotransferase, alanine aminotransferase or bilirubin) by Ghoreishi et al. to predict perioperative mortality in patients undergoing surgical repair for acute type A aortic dissection., Methods: The risk score to predict operative mortality was applied to a large and homogenous validation cohort that consisted of 632 patients undergoing surgery for acute type A aortic dissection in 2 centres. Multivariable regression analysis was performed to determine the impact on survival. Receiver operating characteristics with deduced area under the curve were used to assess the ability to predict perioperative mortality., Results: A total of 632 patients (54% male, mean age 62 ± 14 years) were assigned to 3 different risk groups according to the calculated mortality score [low risk <7 (31.2%), moderate risk 7-20 (36.1%) and high >20 (32.7%)]. Perioperative mortality was 8% in the low-risk group, 10% in the moderate-risk group and 24% in the high-risk group (P < 0.0001). Receiver operating characteristic analysis of this new score revealed an area under the curve of 0.69 with adequate calibration. In addition, multivariable analysis revealed an independet assocation with perioperative mortality (odds ratio 1.509; 95% confidence interval 1.042-2.185). While overall survival differed between the risk groups (P < 0.0001), the score does not serve as an independent predictor of long-term mortality when adjusted for relevant covariates., Conclusions: The external validation process confirmed that a newly proposed risk score offers clinicians a helpful and reliable tool to improve the preoperative risk assessment of acute type A aortic dissection patients based on easily accessible and broadly available laboratory parameters., (© The Author(s) 2021. Published by Oxford University Press on behalf of the European Association for Cardio-Thoracic Surgery. All rights reserved.)

In the expanding field of intestinal organoid research, various protocols for three- and two-dimensional organoid-derived cell cultures exist. Two-dimensional organoid-derived monolayers are used to overcome some limitations of three-dimensional organoid cultures. They are increasingly used also in infection research, to study physiological processes and tissue barrier functions, where easy experimental access of pathogens to the luminal and/or basolateral cell surface is required. This has resulted in an increasing number of publications reporting different protocols and media compositions for organoid manipulation, precluding direct comparisons of research outcomes in some cases. With this in mind, here we describe a protocol aimed at the harmonization of seeding conditions for three-dimensional intestinal organoids of four commonly used research species onto cell culture inserts, to create organoid-derived monolayers that form electrophysiologically tight epithelial barriers. We give an in-depth description of media compositions and culture conditions for creating these monolayers, enabling also the less experienced researchers to obtain reproducible results within a short period of time, and which should simplify the comparison of future studies between labs, but also encourage others to consider these systems as alternative cell culture models in their research. Graphic abstract: Schematic workflow of organoid-derived monolayer generation from intestinal spheroid cultures. ECM, extracellular matrix; ODM, organoid-derived monolayer., Competing Interests: Competing interestsThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The funding bodies had no role in the design, collection, analysis, and interpretation of data, or in writing of this protocol or the previous study., (Copyright © 2022 The Authors; exclusive licensee Bio-protocol LLC.)

Apicomplexan parasites, such as Toxoplasma gondii, are unusual in that each cell contains a single apicoplast, a plastid-like organelle that compartmentalizes enzymes involved in the essential 2C-methyl-D-erythritol 4-phosphate pathway of isoprenoid biosynthesis. The last two enzymatic steps in this organellar pathway require electrons from a redox carrier. However, the small iron-sulfur cluster-containing protein ferredoxin, a likely candidate for this function, has not been investigated in this context. We show here that inducible knockdown of T. gondii ferredoxin results in progressive inhibition of growth and eventual parasite death. Surprisingly, this phenotype is not accompanied by ultrastructural changes in the apicoplast or overall cell morphology. The knockdown of ferredoxin was instead associated with a dramatic decrease in cellular levels of the last two metabolites in isoprenoid biosynthesis, 1-hydroxy-2-methyl-2-(E)- butenyl-4-pyrophosphate, and isomeric dimethylallyl pyrophosphate/isopentenyl pyrophosphate. Ferredoxin depletion was also observed to impair gliding motility, consistent with isoprenoid metabolites being important for dolichol biosynthesis, protein prenylation, and modification of other proteins involved in motility. Significantly, pharmacological inhibition of isoprenoid synthesis of the host cell exacerbated the impact of ferredoxin depletion on parasite replication, suggesting that the slow onset of parasite death after ferredoxin depletion is because of isoprenoid scavenging from the host cell and leading to partial compensation of the depleted parasite metabolites upon ferredoxin knockdown. Overall, these findings show that ferredoxin has an essential physiological function as an electron donor for the 2C-methyl-D-erythritol 4-phosphate pathway and is a potential drug target for apicomplexan parasites., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

The apicomplexan zoonotic parasite Toxoplasma gondii has three infective stages: sporozoites in sporulated oocysts, which are shed in unsporulated form into the environment by infected felids; tissue cysts containing bradyzoites, and fast replicating tachyzoites that are responsible for acute toxoplasmosis. The contribution of oocysts to infections in both humans and animals is understudied despite being highly relevant. Only a few diagnostic antigens have been described to be capable of discriminating which parasite stage has caused an infection. Here we provide an extensive overview of the antigens and serological assays used to detect oocyst-driven infections in humans and animals according to the literature. In addition, we critically discuss the possibility to exploit the increasing knowledge of the T. gondii genome and the various 'omics datasets available, by applying predictive algorithms, for the identification of new oocyst-specific proteins for diagnostic purposes. Finally, we propose a workflow for how such antigens and assays based on them should be evaluated to ensure reproducible and robust results.

Toxoplasmosis is a zoonotic infection contracted through Toxoplasma gondii-contaminated food, soil, or water. Seroprevalence in Germany is high, but estimates of disease incidence are scarce. We investigated incidences for various toxoplasmosis manifestations using anonymized healthcare claims data from Germany for 2011-2016. Patients with a toxoplasmosis diagnosis during the annual observational period were considered incident. The estimated incidence was adjusted to the general population age/sex distribution. We estimated an annual average of 8,047 toxoplasmosis patients in Germany. The average incidence of non-pregnancy-associated toxoplasmosis patients was 9.6/100,000 population. The incidence was highest in 2011, at 10.6 (95% CI 9.4-12.6)/100,000 population, and lowest in 2016, at 8.0 (95% CI 7.0-9.4)/100,000 population. The average incidence of toxoplasmosis during pregnancy was 40.3/100,000 pregnancies. We demonstrate a substantial toxoplasmosis disease burden in Germany. Public health and food safety authorities should implement toxoplasmosis-specific prevention programs.

Purpose: Studies on the occurrence of ocular toxoplasmosis (OT) in a general population are rare. Therefore, we conducted this pilot study to assess whether a nonmydriatic ultra-wide-field (UWF) scanning laser ophthalmoscope (SLO) is suitable for a simple, rapid screening procedure., Methods: The population of this cross-sectional study was randomly recruited from a cohort of hospital-based patients in an urban geriatric hospital. Ophthalmologic evaluation was performed on 201 eyes from 101 participants through nonmydriatic UWF-SLO (Optos Daytona) and assessed for suspicious lesions and other relevant ocular findings. All images were evaluated by two independent examiners. Individuals who presented lesions with a morphological appearance suggestive of OT underwent fundoscopy and serological analysis of Toxoplasma gondii-specific antibodies., Results: The mean age of the study group was 76 years, and 63 (62%) were female. Despite many health restrictions, the SLO examination was carried out easily in this geriatric population. Three participants presented findings by SLO suspicious for T. gondii-related injury. Further clinical examination and serological investigation confirmed the diagnosis, with funduscopic evaluation and positive T. gondii ELISA testing. In addition, a high rate of arterial hypertension and dyslipidemias within the cohort led to a high incidence of vascular changes and age-related fundus findings., Conclusion: In our study, we confirm that UWF-SLO technology is helpful in the rapid detection of peripheral retinal injuries in elderly patients such as OT and may be used as a routine screening tool.

The environmental stage of the apicomplexan Toxoplasma gondii oocyst is vital to its life cycle but largely understudied. Because oocysts are excreted only by infected felids, their availability for research is limited. We report the adaptation of an agarose-based method to immobilize minute amounts of oocysts to perform immunofluorescence assays. Agarose embedding allows high-resolution confocal microscopy imaging of antibodies binding to the oocyst surface as well as unprecedented imaging of intracellular sporocyst structures with Maclura pomifera agglutinin after on-slide permeabilization of the immobilized oocysts. To identify new possible molecules binding to the oocyst surface, we used this method to screen a library of C-type lectin receptor (CLR)-human IgG constant region fusion proteins from the group of related CLRs called the Dectin-1 cluster against oocysts. In addition to CLEC7A that was previously reported to decorate T. gondii oocysts, we present experimental evidence for specific binding of three additional CLRs to the surface of this stage. We discuss how these CLRs, known to be expressed on neutrophils, dendritic cells, or macrophages, could be involved in the early immune response by the host, such as oocyst antigen uptake in the intestine. In conclusion, we present a modified immunofluorescence assay technique that allows material-saving immunofluorescence microscopy with T. gondii oocysts in a higher resolution than previously published, which allowed us to describe three additional CLRs binding specifically to the oocyst surface. IMPORTANCE Knowledge of oocyst biology of Toxoplasma gondii is limited, not the least due to its limited availability. We describe a method that permits us to process minute amounts of oocysts for immunofluorescence microscopy without compromising their structural properties. This method allowed us to visualize internal structures of sporocysts by confocal microscopy in unprecedented quality. Moreover, the method can be used as a low- to medium-throughput method to screen for molecules interacting with oocysts, such as antibodies, or compounds causing structural damage to oocysts (i.e., disinfectants). Using this method, we screened a small library of C-type lectin receptors (CLRs) present on certain immune cells and found three CLRs able to decorate the oocyst wall of T. gondii and which were not known before to bind to oocysts. These tools will allow further study into oocyst wall composition and could also provoke experiments regarding immunological recognition of oocysts., (Copyright © 2021 Fabian et al.)

The small intestinal epithelium is the primary route of infection for many protozoan parasites. Understanding the mechanisms of infection, however, has been hindered due to the lack of appropriate models that recapitulate the complexity of the intestinal epithelium. Here, we describe an in vitro platform using stem cell-derived intestinal organoids established for four species that are important hosts of Apicomplexa and other protozoa in a zoonotic context: human, mouse, pig and chicken. The focus was set to create organoid-derived monolayers (ODMs) using the transwell system amenable for infection studies, and we provide straightforward guidelines for their generation and differentiation from organ-derived intestinal crypts. To this end, we reduced medium variations to an absolute minimum, allowing generation and differentiation of three-dimensional organoids for all four species and the subsequent generation of ODMs. Quantitative RT-PCR, immunolabeling with antibodies against marker proteins as well as transepithelial-electrical resistance (TEER) measurements were used to characterize ODM's integrity and functional state. These experiments show an overall uniform generation of monolayers suitable for Toxoplasma gondii infection, although robustness in terms of generation of stable TEER levels and cell differentiation status varies from species to species. Murine duodenal ODMs were then infected with T. gondii and/or Giardia duodenalis , two parasites that temporarily co-inhabit the intestinal niche but have not been studied previously in cellular co-infection models. T. gondii alone did not alter TEER values, integrity and transcriptional abundance of tight junction components. In contrast, in G. duodenalis -infected ODMs all these parameters were altered and T. gondii had no apparent influence on the G. duodenalis -triggered phenotype. In conclusion, we provide robust protocols for the generation, differentiation and characterization of intestinal organoids and ODMs from four species. We show their applications for comparative studies on parasite-host interactions during the early phase of a T. gondii infection but also its use for co-infections with other relevant intestinal protozoans., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Holthaus, Delgado-Betancourt, Aebischer, Seeber and Klotz.)

Background: Few bead-based multiplex assays have been described that detect antibodies against the protozoan parasite Toxoplasma gondii in large-scale seroepidemiological surveys. Moreover, each multiplex assay has specific variations or limitations, such as the use of truncated or fusion proteins as antigens, potentially masking important epitopes. Consequently, such an assay must be developed by interested groups as none is commercially available., Results: We report the bacterial expression and use of N-terminal fusion-free, soluble, in vivo biotinylated recombinant surface antigens SAG1 and SAG2A for the detection of anti-T. gondii IgG antibodies. The expression system relies on three compatible plasmids. An expression construct produces a fusion of maltose-binding protein with SAG1 (or SAG2A), separated by a TEV protease cleavage site, followed by a peptide sequence recognized by E. coli biotin ligase BirA (AviTag), and a terminal six histidine tag for affinity purification. TEV protease and BirA are encoded on a second plasmid, and their expression leads to proteolytic cleavage of the fusion protein and a single biotinylated lysine within the AviTag by BirA. Correct folding of the parasite proteins is dependent on proper disulfide bonding, which is facilitated by a sulfhydryl oxidase and a protein disulfide isomerase, encoded on the third plasmid. The C-terminal biotinylation allowed the oriented, reproducible coupling of the purified surface antigens to magnetic Luminex beads, requiring only minute amounts of protein per determination. We showed that an N-terminal fusion partner such as maltose-binding protein negatively influenced antibody binding, confirming that access to SAG1's N-terminal epitopes is important for antibody recognition. We validated our bead-based multiplex assay with human sera previously tested with commercial diagnostic assays and found concordance of 98-100% regarding both, sensitivity and specificity, even when only biotinylated SAG1 was used as antigen., Conclusions: Our recombinant in vivo-biotinylated T. gondii antigens offer distinct advantages compared to previously described proteins used in multiplex serological assays for T. gondii. They offer a cheap, specific and sensitive alternative to either parasite lysates or eukaryotic-cell expressed SAG1/SAG2A for BBMA and other formats. The described general expression strategy can also be used for other antigens where oriented immobilization is key for sensitive recognition by antibodies and ligands.

Background: Free-ranging chickens are often infected with Toxoplasma gondii and seroconvert upon infection. This indicates environmental contamination with T. gondii., Methods: Here, we established a bead-based multiplex assay (BBMA) using the Luminex technology for the detection of T. gondii infections in chickens. Recombinant biotinylated T. gondii surface antigen 1 (TgSAG1 bio ) bound to streptavidin-conjugated magnetic Luminex beads served as antigen. Serum antibodies were detected by a fluorophore-coupled secondary antibody. Beads of differing color codes were conjugated with anti-chicken IgY or chicken serum albumin and served for each sample as an internal positive or negative control, respectively. The assay was validated with sera from experimentally and naturally infected chickens. The results were compared to those from reference methods, including other serological tests, PCRs and bioassay in mice., Results: In experimentally infected chickens, the vast majority (98.5%, n = 65/66) of birds tested seropositive in the BBMA. This included all chickens positive by magnetic-capture PCR (100%, n = 45/45). Most, but not all inoculated and TgSAG1 bio -BBMA-positive chickens were also positive in two previously established TgSAG1-ELISAs (TgSAG1-ELISA SL , n = 61/65; or TgSAG1-ELISA SH , n = 60/65), or positive in an immunofluorescence assay (IFAT, n = 64/65) and in a modified agglutination test (MAT, n = 61/65). All non-inoculated control animals (n = 28/28, 100%) tested negative. In naturally exposed chickens, the TgSAG1 bio -BBMA showed a high sensitivity (98.5%; 95% confidence interval, CI: 90.7-99.9%) and specificity (100%; 95% CI: 85.0-100%) relative to a reference standard established using ELISA, IFAT and MAT. Almost all naturally exposed chickens that were positive in bioassay or by PCR tested positive in the TgSAG1 bio -BBMA (93.5%; 95% CI: 77.1-98.9%), while all bioassay- or PCR-negative chickens remained negative (100%; 95% CI: 85.0-100%)., Conclusions: The TgSAG1 bio -BBMA represents a suitable method for the detection of T. gondii infections in chickens with high sensitivity and specificity, which is comparable or even superior to other tests. Since assays based on this methodology allow for the simultaneous analysis of a single biological sample with respect to multiple analytes, the described assay may represent a component in future multiplex assays for broad serological monitoring of poultry and other farm animals for various pathogens.

Toxoplasmosis, while often an asymptomatic parasitic disease in healthy individuals, can cause severe complications in immunocompromised persons and during pregnancy. The most common method to diagnose Toxoplasma gondii infections is the serological determination of antibodies directed against parasite protein antigens. Here we report the use of a bead-based multiplex assay containing a synthetic phosphoglycan portion of the Toxoplasma gondii glycosylphosphatidylinositol (GPI 1 ) for the detection of GPI 1 -specific antibodies in human sera. The glycan was conjugated to beads at the lipid site to retain its natural orientation and its immunogenic groups. We compared the response against GPI 1 with that against the protein antigen SAG1, a common component of commercial serological assays, via the detection of parasite-specific human IgG and IgM antibodies, respectively. The GPI 1 -based test is in excellent agreement with the results for the commercial ELISA, as the ROC analysis of the GPI 1 test shows 97% specificity and 98% sensitivity for the assay. GPI 1 was a more reliable predictor for a parasite-specific IgM response compared to SAG1, indicating that a bead-based multiplex assay using GPI 1 in combination with SAG1 may strengthen Toxoplasma gondii serology, in particular in seroepidemiological studies.

Background: With approximately 30% of the world population infected, Toxoplasma gondii is one of the most widespread pathogenic parasites in both humans and animals and a major problem for health economics in many countries., Methods: This review is based on the findings of individual studies, meta-analyses, and Cochrane Reviews retrieved by a selective literature survey of the Medline and Google Scholar databases., Results: Current data indicate a high rate of Toxoplasma gondii infection in Germany, ranging from 20% to 77% depending on age (95% confidence interval for 18- to 29-year-olds [17.0; 23.1]; for 70- to 79-year-olds [72.7; 80.5]). Male sex, caring for a cat, and a body mass index of 30 or more are independent risk factors for seroconversion. Postnatally acquired (food-related) infec- tion is predominant, but maternal-to-fetal transmission still plays an important role. While most infections are asymptomatic, congenital toxoplasmosis and reactivated Toxoplasma encephalitis in immunosuppressed persons (transplant recipients and others) are sources of considerable morbidity. Toxoplasma gondii infection of the retina is the most common cause of infectious uveitis in Germany. The diagnosis and treatment of this type of parasitic infection are particular to the specific organs involved in the individual patient., Conclusion: Desirable steps for the near future include development of an effective treatment for the cystic stage and identifica- tion of biomarkers to assess the risk of reactivation and predict the disease course.

Toxoplasma gondii is a zoonotic intracellular parasite, able to infect any warm-blooded animal via ingestion of infective stages, either contained in tissue cysts or oocysts released into the environment. While immune responses during infection are well-studied, there is still limited knowledge about the very early infection events in the gut tissue after infection via the oral route. Here we briefly discuss differences in host-specific responses following infection with oocyst-derived sporozoites vs. tissue cyst-derived bradyzoites. A focus is given to innate intestinal defense mechanisms and early immune cell events that precede T. gondii' s dissemination in the host. We propose stem cell-derived intestinal organoids as a model to study early events of natural host-pathogen interaction. These offer several advantages such as live cell imaging and transcriptomic profiling of the earliest invasion processes. We additionally highlight the necessity of an appropriate large animal model reflecting human infection more closely than conventional infection models, to study the roles of dendritic cells and macrophages during early infection.

Toxoplasma gondii is an ubiquitous intracellular protozoan parasite. Mammals and birds are intermediate hosts and felid species are definitive hosts. In most human altered habitats the domestic cat is the predominant definitive host. Current knowledge of T. gondii infection in African ecosystems is limited. This study aimed to assess exposure to T. gondii in wild carnivores in the Serengeti ecosystem in East Africa. Carnivores can be infected by the consumption of tissue cysts when feeding on infected animals and by incidental ingestion of oocysts from environmental contamination. Incidental ingestion should occur regardless of a species' diet whereas the consumption of cysts should increase the chance of infection in carnivorous species. This predicts higher seropositivity in carnivorous than in insectivorous carnivores and lower seropositivity in juvenile carnivores with a long dependency on milk than in adults. We found high seropositivity in carnivorous species: 100% (15 of 15 samples) in adult African lions, 93% (38 of 41 samples) in adult spotted hyenas and one striped hyena sample was positive, whereas all four samples from the insectivorous bat-eared fox were negative. Juvenile hyenas (11 of 19 sera) had significantly lower seropositivity than adults (38 of 41 sera). Long-term monitoring of spotted hyenas revealed no significant difference in seropositivity between two periods (1988-1992 and 2000 to 2016). Identical results were produced in lion and hyena samples by a commercial multi-species ELISA (at serum dilution 1:10) and an in-house ELISA based on a recombinant T. gondii protein (at serum dilution 1:100), making the latter a useful alternative for small amounts of serum. We suggest that diet, age and lifetime range are factors determining seropositivity in carnivores in the Serengeti ecosystem and suggest that the role of small wild felids in the spread of T. gondii in the African ecosystem warrants investigation.

Toxoplasma gondii is an obligate intracellular parasite belonging to the phylum Apicomplexa that infects all warm-blooded animals, including humans. T. gondii can replicate in every nucleated host cell by orchestrating metabolic interactions to derive crucial nutrients. In this review, we summarize the current status of known metabolic interactions of T. gondii with its host cell and discuss open questions and promising experimental approaches that will allow further dissection of the host-parasite interface and discovery of ways to efficiently target both tachyzoite and bradyzoite forms of T. gondii , which are associated with acute and chronic infection, respectively., Competing Interests: No competing interests were disclosed.No competing interests were disclosed.No competing interests were disclosed.

Rodent species like Myodes glareolus and Microtus spp. are natural reservoirs for many zoonotic pathogens causing human diseases and are gaining increasing interest in the field of eco-immunology as candidate animal models. Despite their importance the lack of immunological reagents has hampered research in these animal species. Here we report the recombinant production and functional characterization of IFN-γ, a central mediator of host's innate and adaptive immune responses, from the bank vole M. glareolus. Soluble dimeric recMgIFN-γ was purified in high yield from Escherichia coli. Its activity on M. glareolus and Microtus arvalis kidney cell lines was assessed by immunofluorescent detection of nuclear translocation and phosphorylation of the transcription factor STAT1. RecMgIFN-γ also induced expression of an IFN-γ-regulated innate immunity gene. Inhibition of vesicular stomatitis virus replication in vole cells upon recMgIFN-γ treatment provided further evidence of its biological activity. Finally, we established a recMgIFN-γ-responsive bank vole reporter cell line that allows the sensitive titration of the cytokine activity via a bioluminescence reporter assay. Taken together, we report valuable tools for future investigations on the immune response against zoonotic pathogens in their natural animal hosts, which might foster the development of novel animal models.