Cryogenic electron tomography reveals novel structures in the apical complex of Plasmodium falciparum.

Intracellular infectious agents, like the malaria parasite, Plasmodium falciparum, face the daunting challenge of how to invade a host cell. This problem may be even harder when the host cell in question is the enucleated red blood cell, which lacks the host machinery co-opted by many pathogens for internalization. Evolution has provided P. falciparum and related single-celled parasites within the phylum Apicomplexa with a collection of organelles at their apical end that mediate invasion. This apical complex includes at least two sets of secretory organelles, micronemes and rhoptries, and several structural features like apical rings and a putative pore through which proteins may be introduced into the host cell during invasion. We perform cryogenic electron tomography (cryo-ET) equipped with Volta Phase Plate on isolated and vitrified merozoites to visualize the apical machinery. Through tomographic reconstruction of cellular compartments, we see new details of known structures like the rhoptry tip interacting directly with a rosette resembling the recently described rhoptry secretory apparatus (RSA), or with an apical vesicle docked beneath the RSA. Subtomogram averaging reveals that the apical rings have a fixed number of repeating units, each of which is similar in overall size and shape to the units in the apical rings of tachyzoites of Toxoplasma gondii. Comparison of these polar rings in Plasmodium and Toxoplasma parasites also reveals them to have a structurally conserved assembly pattern. These results provide new insight into the essential and structurally conserved features of this remarkable machinery used by apicomplexan parasites to invade their respective host cells. IMPORTANCE: Malaria is an infectious disease caused by parasites of the genus Plasmodium and is a leading cause of morbidity and mortality globally. Upon infection, Plasmodium parasites invade and replicate in red blood cells, where they are largely protected from the immune system. To enter host cells, the parasites employ a specialized apparatus at their anterior end. In this study, advanced imaging techniques like cryogenic electron tomography (cryo-ET) and Volta Phase Plate enable unprecedented visualization of whole Plasmodium falciparum merozoites, revealing previously unknown structural details of their invasion machinery. Key findings include new insights into the structural conservation of apical rings shared between Plasmodium and its apicomplexan cousin, Toxoplasma. These discoveries shed light on the essential and conserved elements of the invasion machinery used by these pathogens. Moreover, the research provides a foundation for understanding the molecular mechanisms underlying parasite-host interactions, potentially informing strategies for combating diseases caused by apicomplexan parasites.

Plasmodium falciparum exploits CD44 as a coreceptor for erythrocyte invasion.

The malaria parasite Plasmodium falciparum invades and replicates asexually within human erythrocytes. CD44 expressed on erythrocytes was previously identified as an important host factor for P falciparum infection through a forward genetic screen, but little is known about its regulation or function in these cells, nor how it may be used by the parasite. We found that CD44 can be efficiently deleted from primary human hematopoietic stem cells using CRISPR/Cas9 genome editing, and that the efficiency of ex vivo erythropoiesis to enucleated cultured red blood cells (cRBCs) is not affected by lack of CD44. However, the rate of P falciparum invasion was reduced in CD44-null cRBCs relative to isogenic wild-type control cells, validating CD44 as an important host factor for this parasite. We identified 2 P falciparum invasion ligands as binding partners for CD44, erythrocyte binding antigen 175 (EBA-175) and EBA-140 and demonstrated that their ability to bind to human erythrocytes relies primarily on their canonical receptors, glycophorin A and glycophorin C, respectively. We further show that EBA-175 induces phosphorylation of erythrocyte cytoskeletal proteins in a CD44-dependent manner. Our findings support a model in which P falciparum exploits CD44 as a coreceptor during invasion of human erythrocytes, stimulating CD44-dependent phosphorylation of host cytoskeletal proteins that alter host cell deformability and facilitate parasite entry.

Plasmodium falciparum infection of human erythroblasts induces transcriptional changes associated with dyserythropoiesis.

During development down the erythroid lineage, hematopoietic stem cells undergo dramatic changes to cellular morphology and function in response to a complex and tightly regulated program of gene expression. In malaria infection, Plasmodium spp . parasites accumulate in the bone marrow parenchyma, and emerging evidence suggests erythroblastic islands are a protective site for parasite development into gametocytes. While it has been observed that Plasmodium falciparum infection of late-stage erythroblasts can delay terminal erythroid differentiation and enucleation, the mechanism(s) underlying this phenomenon are unknown. Here, we apply RNA-seq after fluorescence-activated cell sorting (FACS) of infected erythroblasts to identify transcriptional responses to direct and indirect interaction with Plasmodium falciparum . Four developmental stages of erythroid cells were analyzed: proerythroblast, basophilic erythroblast, polychromatic erythroblast, and orthochromatic erythroblast. We found extensive transcriptional changes in infected erythroblasts compared to uninfected cells in the same culture, including dysregulation of genes involved in erythroid proliferation and developmental processes. Whereas some indicators of cellular oxidative and proteotoxic stress were common across all stages of erythropoiesis, many responses were specific to cellular processes associated with developmental stage. Together, our results evidence multiple possible avenues by which parasite infection can induce dyserythropoiesis at specific points along the erythroid continuum, advancing our understanding of the molecular determinants of malaria anemia. Key Points: Erythroblasts at different stages of differentiation have distinct responses to infection by Plasmodium falciparum . P. falciparum infection of erythroblasts alters expression of genes related to oxidative and proteotoxic stress and erythroid development.

Plasmodium falciparum infection of human erythroblasts induces transcriptional changes associated with dyserythropoiesis.

During development down the erythroid lineage, hematopoietic stem cells undergo dramatic changes to cellular morphology and function in response to a complex and tightly regulated program of gene expression. In malaria infection, Plasmodium spp parasites accumulate in the bone marrow parenchyma, and emerging evidence suggests erythroblastic islands are a protective site for parasite development into gametocytes. Although it has been observed that Plasmodium falciparum infection in late-stage erythroblasts can delay terminal erythroid differentiation and enucleation, the mechanism(s) underlying this phenomenon are unknown. Here, we apply RNA sequencing after fluorescence-activated cell sorting of infected erythroblasts to identify transcriptional responses to direct and indirect interaction with P falciparum. Four developmental stages of erythroid cells were analyzed: proerythroblast, basophilic erythroblast, polychromatic erythroblast, and orthochromatic erythroblast. We found extensive transcriptional changes in infected erythroblasts compared with that in uninfected cells in the same culture, including dysregulation of genes involved in erythroid proliferation and developmental processes. Although some indicators of cellular oxidative and proteotoxic stress were common across all stages of erythropoiesis, many responses were specific to cellular processes associated with developmental stage. Together, our results evidence multiple possible avenues by which parasite infection can induce dyserythropoiesis at specific points along the erythroid continuum, advancing our understanding of the molecular determinants of malaria anemia.

Plasmodium falciparum exploits CD44 as a co-receptor for erythrocyte invasion.

The malaria parasite Plasmodium falciparum invades and replicates asexually within human erythrocytes. CD44 expressed on erythrocytes was previously identified as an important host factor for P. falciparum infection through a forward genetic screen, but little is known about its regulation or function in these cells, nor how it may be utilized by the parasite. We found that CD44 can be efficiently deleted from primary human hematopoietic stem cells using CRISPR/Cas9 genome editing, and that the efficiency of ex-vivo erythropoiesis to enucleated cultured red blood cells (cRBCs) is not impacted by lack of CD44. However, the rate of P. falciparum invasion was substantially reduced in CD44-null cRBCs relative to isogenic wild-type (WT) control cells, validating CD44 as an important host factor for this parasite. We identified two P. falciparum invasion ligands as binding partners for CD44, Erythrocyte Binding Antigen-175 (EBA-175) and EBA-140, and demonstrated that their ability to bind to human erythrocytes relies primarily on their canonical receptors-glycophorin A and glycophorin C, respectively. We further show that EBA-175 induces phosphorylation of erythrocyte cytoskeletal proteins in a CD44-dependent manner. Our findings support a model where P. falciparum exploits CD44 as a co-receptor during invasion of human erythrocytes, stimulating CD44-dependent phosphorylation of host cytoskeletal proteins that alter host cell deformability and facilitate parasite entry.

Antigenic diversity in malaria parasites is maintained on extrachromosomal DNA.

Sequence variation among antigenic var genes enables Plasmodium falciparum malaria parasites to evade host immunity. Using long sequence reads from haploid clones from a mutation accumulation experiment, we detect var diversity inconsistent with simple chromosomal inheritance. We discover putatively circular DNA that is strongly enriched for var genes, which exist in multiple alleles per locus separated by recombination and indel events. Extrachromosomal DNA likely contributes to rapid antigenic diversification in P. falciparum.

Cryo-electron tomography with mixed-scale dense neural networks reveals key steps in deployment of Toxoplasma invasion machinery.

Host cell invasion by intracellular, eukaryotic parasites within the phylum Apicomplexa is a remarkable and active process involving the coordinated action of apical organelles and other structures. To date, capturing how these structures interact during invasion has been difficult to observe in detail. Here, we used cryogenic electron tomography to image the apical complex of Toxoplasma gondii tachyzoites under conditions that mimic resting parasites and those primed to invade through stimulation with calcium ionophore. Through the application of mixed-scale dense networks for image processing, we developed a highly efficient pipeline for annotation of tomograms, enabling us to identify and extract densities of relevant subcellular organelles and accurately analyze features in 3-D. The results reveal a dramatic change in the shape of the anteriorly located apical vesicle upon its apparent fusion with a rhoptry that occurs only in the stimulated parasites. We also present information indicating that this vesicle originates from the vesicles that parallel the intraconoidal microtubules and that the latter two structures are linked by a novel tether. We show that a rosette structure previously proposed to be involved in rhoptry secretion is associated with apical vesicles beyond just the most anterior one. This result, suggesting multiple vesicles are primed to enable rhoptry secretion, may shed light on the mechanisms Toxoplasma employs to enable repeated invasion attempts. Using the same approach, we examine Plasmodium falciparum merozoites and show that they too possess an apical vesicle just beneath a rosette, demonstrating evolutionary conservation of this overall subcellular organization.

Erythrocyte-Plasmodium interactions: genetic manipulation of the erythroid lineage.

Targeting critical host factors is an emerging concept in the treatment of infectious diseases. As obligate pathogens of erythrocytes, the Plasmodium spp. parasites that cause malaria must exploit erythroid host factors for their survival. However, our understanding of this important aspect of the malaria lifecycle is limited, in part because erythrocytes are enucleated cells that lack a nucleus and DNA, rendering them genetically intractable. Recent advances in genetic analysis of the erythroid lineage using small-hairpin RNAs and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated nuclease 9 (Cas9) in red-blood cells derived from stem cells have generated new insights into the functions of several candidate host factors for Plasmodium parasites. Along with efforts in other hematopoietic cells, these advances have also laid a strong foundation for genetic screens to identify novel erythrocyte host factors for malaria.

Automated Recognition of Plasmodium falciparum Parasites from Portable Blood Levitation Imaging.

In many malaria-endemic regions, current detection tools are inadequate in diagnostic accuracy and accessibility. To meet the need for direct, phenotypic, and automated malaria parasite detection in field settings, a portable platform to process, image, and analyze whole blood to detect Plasmodium falciparum parasites, is developed. The liberated parasites from lysed red blood cells suspended in a magnetic field are accurately detected using this cellphone-interfaced, battery-operated imaging platform. A validation study is conducted at Ugandan clinics, processing 45 malaria-negative and 36 malaria-positive clinical samples without external infrastructure. Texture and morphology features are extracted from the sample images, and a random forest classifier is trained to assess infection status, achieving 100% sensitivity and 91% specificity against gold-standard measurements (microscopy and polymerase chain reaction), and limit of detection of 31 parasites per µL. This rapid and user-friendly platform enables portable parasite detection and can support malaria diagnostics, surveillance, and research in resource-constrained environments.

Uncovering a Cryptic Site of Malaria Pathogenesis: Models to Study Interactions Between Plasmodium and the Bone Marrow.

The bone marrow is a critical site of host-pathogen interactions in malaria infection. The discovery of Plasmodium asexual and transmission stages in the bone marrow has renewed interest in the tissue as a niche for cellular development of both host and parasite. Despite its importance, bone marrow in malaria infection remains largely unexplored due to the challenge of modeling the complex hematopoietic environment in vitro. Advancements in modeling human erythropoiesis ex-vivo from primary human hematopoietic stem and progenitor cells provide a foothold to study the host-parasite interactions occurring in this understudied site of malaria pathogenesis. This review focuses on current in vitro methods to recapitulate and assess bone marrow erythropoiesis and their potential applications in the malaria field. We summarize recent studies that leveraged ex-vivo erythropoiesis to shed light on gametocyte development in nucleated erythroid stem cells and begin to characterize host cell responses to Plasmodium infection in the hematopoietic niche. Such models hold potential to elucidate mechanisms of disordered erythropoiesis, an underlying contributor to malaria anemia, as well as understand the biological determinants of parasite sexual conversion. This review compares the advantages and limitations of the ex-vivo erythropoiesis approach with those of in vivo human and animal studies of the hematopoietic niche in malaria infection. We highlight the need for studies that apply single cell analyses to this complex system and incorporate physical and cellular components of the bone marrow that may influence erythropoiesis and parasite development.

Talimogene laherparepvec resulting in near-complete response in a patient with treatment-refractory Merkel cell carcinoma.

Merkel cell carcinoma (MCC) is a rare and aggressive cutaneous tumour of neuroendocrine cell origin, which can grow rapidly and metastasise early. Localised disease is treated with surgery and radiotherapy. Disease that reaches a more advanced stage can be treated with a variety of different treatment modalities including surgery, radiotherapy, chemotherapy, radionuclide therapy, immunotherapy, and intralesional therapy. We report a case of a patient who had exhausted all local and systemic treatment options and who subsequently had an exceptional response to intralesional injection of Talimogene laherparepvec (TVEC).

Revisiting the malaria hypothesis: accounting for polygenicity and pleiotropy.

The malaria hypothesis predicts local, balancing selection of deleterious alleles that confer strong protection from malaria. Three protective variants, recently discovered in red cell genes, are indeed more common in African than European populations. Still, up to 89% of the heritability of severe malaria is attributed to many genome-wide loci with individually small effects. Recent analyses of hundreds of genome-wide association studies (GWAS) in humans suggest that most functional, polygenic variation is pleiotropic for multiple traits. Interestingly, GWAS alleles and red cell traits associated with small reductions in malaria risk are not enriched in African populations. We propose that other selective and neutral forces, in addition to malaria prevalence, explain the global distribution of most genetic variation impacting malaria risk.

Mitochondria-Rich Extracellular Vesicles Rescue Patient-Specific Cardiomyocytes From Doxorubicin Injury: Insights Into the SENECA Trial.

BACKGROUND: Anthracycline-induced cardiomyopathy (AIC) is a significant source of morbidity and mortality in cancer survivors. The role of mesenchymal stem cells (MSCs) in treating AIC was evaluated in the SENECA trial, a Phase 1 National Heart, Lung, and Blood Institute-sponsored study, but the mechanisms underpinning efficacy in human tissue need clarification. OBJECTIVES: The purpose of this study was to perform an in vitro clinical trial evaluating the efficacy and putative mechanisms of SENECA trial-specific MSCs in treating doxorubicin (DOX) injury, using patient-specific induced pluripotent stem cell-derived cardiomyocytes (iCMs) generated from SENECA patients. METHODS: Patient-specific iCMs were injured with 1 µmol/L DOX for 24 hours, treated with extracellular vesicles (EVs) from MSCs by either coculture or direct incubation and then assessed for viability and markers of improved cellular physiology. MSC-derived EVs were separated into large extracellular vesicles (L-EVs) (>200 nm) and small EVs (<220nm) using a novel filtration system. RESULTS: iCMs cocultured with MSCs in a transwell system demonstrated improved iCM viability and attenuated apoptosis. L-EVs but not small EVs recapitulated this therapeutic effect. L-EVs were found to be enriched in mitochondria, which were shown to be taken up by iCMs. iCMs treated with L-EVs demonstrated improved contractility, reactive oxygen species production, ATP production, and mitochondrial biogenesis. Inhibiting L-EV mitochondrial function with 1-methyl-4-phenylpyridinium attenuated efficacy. CONCLUSIONS: L-EV-mediated mitochondrial transfer mitigates DOX injury in patient-specific iCMs. Although SENECA was not designed to test MSC efficacy, consistent tendencies toward a positive effect were observed across endpoints. Our results suggest a mechanism by which MSCs may improve cardiovascular performance in AIC independent of regeneration, which could inform future trial design evaluating the therapeutic potential of MSCs.

Common host variation drives malaria parasite fitness in healthy human red cells.

The replication of Plasmodium falciparum parasites within red blood cells (RBCs) causes severe disease in humans, especially in Africa. Deleterious alleles like hemoglobin S are well-known to confer strong resistance to malaria, but the effects of common RBC variation are largely undetermined. Here, we collected fresh blood samples from 121 healthy donors, most with African ancestry, and performed exome sequencing, detailed RBC phenotyping, and parasite fitness assays. Over one-third of healthy donors unknowingly carried alleles for G6PD deficiency or hemoglobinopathies, which were associated with characteristic RBC phenotypes. Among non-carriers alone, variation in RBC hydration, membrane deformability, and volume was strongly associated with P. falciparum growth rate. Common genetic variants in PIEZO1, SPTA1/SPTB, and several P. falciparum invasion receptors were also associated with parasite growth rate. Interestingly, we observed little or negative evidence for divergent selection on non-pathogenic RBC variation between Africans and Europeans. These findings suggest a model in which globally widespread variation in a moderate number of genes and phenotypes modulates P. falciparum fitness in RBCs.

Multiparametric biophysical profiling of red blood cells in malaria infection.

Biophysical separation promises label-free, less-invasive methods to manipulate the diverse properties of live cells, such as density, magnetic susceptibility, and morphological characteristics. However, some cellular changes are so minute that they are undetectable by current methods. We developed a multiparametric cell-separation approach to profile cells with simultaneously changing density and magnetic susceptibility. We demonstrated this approach with the natural biophysical phenomenon of Plasmodium falciparum infection, which modifies its host erythrocyte by simultaneously decreasing density and increasing magnetic susceptibility. Current approaches have used these properties separately to isolate later-stage infected cells, but not in combination. We present biophysical separation of infected erythrocytes by balancing gravitational and magnetic forces to differentiate infected cell stages, including early stages for the first time, using magnetic levitation. We quantified height distributions of erythrocyte populations-27 ring-stage synchronized samples and 35 uninfected controls-and quantified their unique biophysical signatures. This platform can thus enable multidimensional biophysical measurements on unique cell types.

Erythrocyte CD55 mediates the internalization of Plasmodium falciparum parasites.

Invasion of human erythrocytes by the malaria parasite Plasmodium falciparum is a multi-step process. Previously, a forward genetic screen for P. falciparum host factors identified erythrocyte CD55 as essential for invasion, but its specific role and how it interfaces with the other factors that mediate this complex process are unknown. Using CRISPR-Cas9 editing, antibody-based inhibition, and live cell imaging, here we show that CD55 is specifically required for parasite internalization. Pre-invasion kinetics, erythrocyte deformability, and echinocytosis were not influenced by CD55, but entry was inhibited when CD55 was blocked or absent. Visualization of parasites attached to CD55-null erythrocytes points to a role for CD55 in stability and/or progression of the moving junction. Our findings demonstrate that CD55 acts after discharge of the parasite's rhoptry organelles, and plays a unique role relative to all other invasion receptors. As the requirement for CD55 is strain-transcendent, these results suggest that CD55 or its interacting partners may hold potential as therapeutic targets for malaria.

Mitochondria-Rich Extracellular Vesicles From Autologous Stem Cell-Derived Cardiomyocytes Restore Energetics of Ischemic Myocardium.

BACKGROUND: Mitochondrial dysfunction results in an imbalance between energy supply and demand in a failing heart. An innovative therapy that targets the intracellular bioenergetics directly through mitochondria transfer may be necessary. OBJECTIVES: The purpose of this study was to establish a preclinical proof-of-concept that extracellular vesicle (EV)-mediated transfer of autologous mitochondria and their related energy source enhance cardiac function through restoration of myocardial bioenergetics. METHODS: Human-induced pluripotent stem cell-derived cardiomyocytes (iCMs) were employed. iCM-conditioned medium was ultracentrifuged to collect mitochondria-rich EVs (M-EVs). Therapeutic effects of M-EVs were investigated using in vivo murine myocardial infarction (MI) model. RESULTS: Electron microscopy revealed healthy-shaped mitochondria inside M-EVs. Confocal microscopy showed that M-EV-derived mitochondria were transferred into the recipient iCMs and fused with their endogenous mitochondrial networks. Treatment with 1.0 × 108/ml M-EVs significantly restored the intracellular adenosine triphosphate production and improved contractile profiles of hypoxia-injured iCMs as early as 3 h after treatment. In contrast, isolated mitochondria that contained 300× more mitochondrial proteins than 1.0 × 108/ml M-EVs showed no effect after 24 h. M-EVs contained mitochondrial biogenesis-related messenger ribonucleic acids, including proliferator-activated receptor γ coactivator-1α, which on transfer activated mitochondrial biogenesis in the recipient iCMs at 24 h after treatment. Finally, intramyocardial injection of 1.0 × 108 M-EVs demonstrated significantly improved post-MI cardiac function through restoration of bioenergetics and mitochondrial biogenesis. CONCLUSIONS: M-EVs facilitated immediate transfer of their mitochondrial and nonmitochondrial cargos, contributing to improved intracellular energetics in vitro. Intramyocardial injection of M-EVs enhanced post-MI cardiac function in vivo. This therapy can be developed as a novel, precision therapeutic for mitochondria-related diseases including heart failure.

Voluntary folic acid fortification levels of food staples in Ireland continue to decline: further implications for passive folic acid intakes?

BACKGROUND: Ireland previously had widespread voluntary fortification but research at Dublin City University carried out in 2014 by our research group demonstrated a major decline in the number of food staples fortified with folic acid in Irish supermarkets over the previous 10 years. The aim of the study was to repeat the audit conducted 3 years ago to compare the levels of folic acid fortification of foodstuffs over this time-frame. METHODS: Over a period of 8-weeks between June and August 2017, the nutrition labels of all foodstuffs that might typically be fortified with micronutrients for sale in the supermarkets with the majority of market share in Ireland were examined. The amount of added folic acid detailed on the label was compared with those captured in 2014. RESULTS: In total, 1081 products with added micronutrients were examined. In percentage terms, there has been a decline of Folic Acid (FA) fortified products within the food groups-spreads, breads, cereals, cereal snacks, milks, fruit juices, yogurts/yogurt drinks and energy drinks since 2014. DISCUSSION: The number of food staples fortified with FA continues to decline demonstrating that voluntary fortification in Ireland is no longer an effective measure for passively augmenting the folic acid levels of consumers.

A common polymorphism in the mechanosensitive ion channel PIEZO1 is associated with protection from severe malaria in humans.

Malaria caused by the apicomplexan parasite Plasmodium falciparum has served as a strong evolutionary force throughout human history, selecting for red blood cell polymorphisms that confer innate protection against severe disease. Recently, gain-of-function mutations in the mechanosensitive ion channel PIEZO1 were shown to ameliorate Plasmodium parasite growth, blood-brain barrier dysfunction, and mortality in a mouse model of malaria. In humans, the gain-of-function allele PIEZO1 E756del is highly prevalent and enriched in Africans, raising the possibility that it is under positive selection due to malaria. Here we used a case-control study design to test for an association between PIEZO1 E756del and malaria severity among children in Gabon. We found that the E756del variant is strongly associated with protection against severe malaria in heterozygotes. In subjects with sickle cell trait, heterozygosity for PIEZO1 E756del did not confer additive protection and homozygosity was associated with an elevated risk of severe disease, suggesting an epistatic relationship between hemoglobin S and PIEZO1 E756del. Using donor blood samples, we show that red cells heterozygous for PIEZO1 E756del are not dehydrated and can support the intracellular growth of P. falciparum similar to wild-type cells. However, surface expression of the P. falciparum virulence protein PfEMP-1 was significantly reduced in infected cells heterozygous for PIEZO1 756del, a phenomenon that has been observed with other protective polymorphisms, such as hemoglobin C. Our findings demonstrate that PIEZO1 is an important innate determinant of malaria susceptibility in humans and suggest that the mechanism of protection may be related to impaired export of P. falciparum virulence proteins.

Are Postoperative Clinical Outcomes Influenced by Length of Stay in the Postanesthesia Care Unit?

PURPOSE: To compare clinical outcomes of patients who required a prolonged length of stay in the postanesthesia care unit (PACU) with a control group. DESIGN: A single-center purposive-sampled retrospective medical record and database audit. METHODS: Patients with prolonged PACU stays were compared to a group of patients whose stay was less than median for outcome measures: rapid response team (RRT) activation, cardiac arrest, unanticipated intensive care unit admissions, and survival to discharge. FINDINGS: A total of 1,867 patients were included in the analysis (n = 931 prolonged stay and n = 933 control group). Prolonged stay in PACU was higher among patients who were older, had higher American Society of Anesthesiologist score, and were discharged to wards during the afternoon or late nursing shift. RRT activation after discharge from PACU occurred in more patients in the study group compared with the control group (7% vs 1%, respectively). There were no cardiac arrests recorded in either group within the 24 hours after PACU discharge period. CONCLUSIONS: Prolonged stay in the PACU for 2 or more hours because of clinical reasons appears to be associated with a higher incidence of clinical deterioration in the ward setting requiring RRT intervention within 24 hours after discharge from PACU.