mRNA vaccine against malaria tailored for liver-resident memory T cells.

Malaria is caused by Plasmodium species transmitted by Anopheles mosquitoes. Following a mosquito bite, Plasmodium sporozoites migrate from skin to liver, where extensive replication occurs, emerging later as merozoites that can infect red blood cells and cause symptoms of disease. As liver tissue-resident memory T cells (Trm cells) have recently been shown to control liver-stage infections, we embarked on a messenger RNA (mRNA)-based vaccine strategy to induce liver Trm cells to prevent malaria. Although a standard mRNA vaccine was unable to generate liver Trm or protect against challenge with Plasmodium berghei sporozoites in mice, addition of an agonist that recruits T cell help from type I natural killer T cells under mRNA-vaccination conditions resulted in significant generation of liver Trm cells and effective protection. Moreover, whereas previous exposure of mice to blood-stage infection impaired traditional vaccines based on attenuated sporozoites, mRNA vaccination was unaffected, underlining the potential for such a rational mRNA-based strategy in malaria-endemic regions.

The CD8+ T cell tolerance checkpoint triggers a distinct differentiation state defined by protein translation defects.

Peripheral CD8+ T cell tolerance is a checkpoint in both autoimmune disease and anti-cancer immunity. Despite its importance, the relationship between tolerance-induced states and other CD8+ T cell differentiation states remains unclear. Using flow cytometric phenotyping, single-cell RNA sequencing (scRNA-seq), and chromatin accessibility profiling, we demonstrated that in vivo peripheral tolerance to a self-antigen triggered a fundamentally distinct differentiation state separate from exhaustion, memory, and functional effector cells but analogous to cells defectively primed against tumors. Tolerant cells diverged early and progressively from effector cells, adopting a transcriptionally and epigenetically distinct state within 60 h of antigen encounter. Breaching tolerance required the synergistic actions of strong T cell receptor (TCR) signaling and inflammation, which cooperatively induced gene modules that enhanced protein translation. Weak TCR signaling during bystander infection failed to breach tolerance due to the uncoupling of effector gene expression from protein translation. Thus, tolerance engages a distinct differentiation trajectory enforced by protein translation defects.

FOXO1 enhances CAR T cell stemness, metabolic fitness and efficacy.

Chimeric antigen receptor (CAR) T cell therapy has transformed the treatment of haematological malignancies such as acute lymphoblastic leukaemia, B cell lymphoma and multiple myeloma1-4, but the efficacy of CAR T cell therapy in solid tumours has been limited5. This is owing to a number of factors, including the immunosuppressive tumour microenvironment that gives rise to poorly persisting and metabolically dysfunctional T cells. Analysis of anti-CD19 CAR T cells used clinically has shown that positive treatment outcomes are associated with a more 'stem-like' phenotype and increased mitochondrial mass6-8. We therefore sought to identify transcription factors that could enhance CAR T cell fitness and efficacy against solid tumours. Here we show that overexpression of FOXO1 promotes a stem-like phenotype in CAR T cells derived from either healthy human donors or patients, which correlates with improved mitochondrial fitness, persistence and therapeutic efficacy in vivo. This work thus reveals an engineering approach to genetically enforce a favourable metabolic phenotype that has high translational potential to improve the efficacy of CAR T cells against solid tumours.

Primary thromboprophylaxis in cancer outpatients - real-world evidence.

INTRODUCTION: Cancer-associated thrombosis (CAT) is a significant concern among patients with malignant diseases, leading to increased mortality. While current guidelines recommend primary thromboprophylaxis for venous thromboembolism (VTE) in medium-to-high-risk outpatients, this practice remains controversial. A better understanding of primary thromboprophylaxis is crucial, yet there is a lack of Real-World Evidence (RWE) in Portugal. AIMS: This RWE study aimed to elucidate primary thromboprophylaxis practices among cancer outpatients in Portugal. METHODS: A five-year observational multicentric study in eight Portuguese health institutions enrolled 124 adult cancer outpatients under primary thromboprophylaxis for VTE. The endpoints were CAT, bleeding, cancer progression and death. RESULTS: High thrombotic risk tumours were prevalent, with 57% (71) of the patients presenting with pancreatic and gastric cancers. Regarding primary thromboprophylaxis, 55% (68) received Low-Molecular-Weight Heparin (LMWH). VTE was presented in 11% (14) of the patients and major bleeding in 2% (2). Vascular compression, elevated D-dimer and previous VTE were significantly associated with VTE occurrence under primary thromboprophylaxis. The Onkotev model was shown to be the best risk assessment model (RAM) in this population (p = 0.007). CAT patients exhibited a lower progression-free survival than non-CAT patients (p = 0.021), while thrombosis did not influence overall survival (p = 0.542). CONCLUSION: Primary thromboprophylaxis in medium-to-high-risk cancer outpatients is a safe and effective practice in real-world settings. This study is the first Portuguese RWE on primary thromboprophylaxis, highlighting evidence for improving prophylactic strategies in this population.

Endothelial injury in COVID-19 and septic patients.

Systemic inflammatory response, as observed in sepsis and severe COVID-19, may lead to endothelial damage. Therefore, we aim to compare the extent of endothelial injury and its relationship to inflammation in both diseases. We included patients diagnosed with sepsis (SEPSIS group, n = 21), mild COVID-19 (MILD group, n = 31), and severe COVID-19 (SEVERE group, n = 24). Clinical and routine laboratory data were obtained, circulating cytokines (INF-γ, TNF-α, and IL-10) and endothelial injury markers (E-Selectin, Tissue Factor (TF) and von Willebrand factor (vWF)) were measured. Compared to the SEPSIS group, patients with severe COVID-19 present similar clinical and laboratory data, except for lower circulating IL-10 and E-Selectin levels. Compared to the MILD group, patients in the SEVERE group showed higher levels of TNF-α, IL-10, and TF. There was no clear relationship between cytokines and endothelial injury markers among the three studied groups; however, in SEVERE COVID-19 patients, there is a positive relationship between INF-γ with TF and a negative relationship between IL-10 and vWF. In conclusion, COVID-19 and septic patients have a similar pattern of cytokines and endothelial dysfunction markers. These findings highlight the importance of endothelium dysfunction in COVID-19 and suggest that endothelium should be better evaluated as a therapeutic target for the disease.

Hierarchical evaluation of histology and p16-labeling can improve the risk assessment on cervical intraepithelial neoplasia progression.

OBJECTIVE: High-grade cervical lesions (HSIL) are associated with the presence of high-risk HPV types, tissue expression of p16, and increased chance of malignant progression, requiring surgical intervention. To improve risk evaluation, we assessed the discriminatory power of the histological findings associated with p16 immunohistochemistry (IHC) staining to classify the low-grade cervical lesion (LSIL) and HSIL. METHODS: We collected cervical biopsies from colposcopy-visible lesions and non-affected tissue (adjacent to the lesions) of 62 Brazilian women and labeled them with anti-p16 antibodies. In addition to the observational pattern and labeling to define the latent classes (affected vs. non-affected), a computational tool was used for semi-quantitative analysis of p16 expression. The intensity of staining of the nucleus or cytoplasm was captured using the Gimp 2.10 software. ROC curves were used to determine cutoff values for p16 expression in patients classified as LSIL and HSIL by latent class statistics for each labeling stratum. RESULTS: p16 nuclear labeling showed the best sensitivity and specificity to discriminate LSIL with low p16 expression (62%) and HSIL with high p16 expression (37%). Many patients whose lesions had intermediate levels of p16 nuclear staining were subsequently stratified according to the expression of p16 in the cytoplasm, indicating that five of 21 LSIL were at risk of progression, and 13 of 41 HSIL at risk of regression. CONCLUSIONS: We suggest a hierarchical analysis, with histology at the first level, followed by a labeling analysis in the nucleus and then in the cytoplasm to increase the accuracy of the HPV cervical lesion stratification.

Validation of blood gas analysis in arteriovenous fistula for hemodialysis.

INTRODUCTION: Arterial blood gas analysis is a minimally invasive technique used in our daily practice but is not a complication free technique. The aim of this study was to validate results from blood gas analysis obtained from the arteriovenous fistula (AVF)/graft as a surrogate marker of the arterial blood gas analysis. METHODS: A prospective observational study was made in 45 patients. We performed arterial and AVF/graft blood gas analysis and results were compared by a paired sample t Student test. RESULTS: Most of our subjects was male (68.9%) and the mean age was 67 years (±14). Hemodialysis vintage was 63 months (±66), and vascular access age was 62 months (±56). The more prevalent vascular access was left radiocephalic AVF (n = 16; 35.6%) and the main puncture artery was right radial artery (n = 27; 60.0%). There were no statistically significant differences between the samples collected. CONCLUSIONS: Our results suggest a possible alternative of arterial blood gas analysis in AVF/graft for hemodialysis patients. This could result in making an uncomfortable procedure almost painless and reducing complications. Future research should take place to include anatomical characteristics of the AVF or the circulation of recirculation.

Lower prevalence of congenital cytomegalovirus infection in Portugal: possible impact of COVID-19 lockdown?

Cytomegalovirus (CMV) is the most frequent cause of congenital infection all over the world. Its prevalence ranges from 0.2 to 2.2%. Transmission from children to their pregnant mothers is a well-known risk factor, particularly if they attend a childcare centre. This study aims to compare the prevalence of CMV congenital infection (CMV_CI) in Portugal (Lisbon) between two studies, performed respectively in 2019 and 2020. In the 2019 study, performed in two hospitals, we found a 0.67% CMV_CI prevalence, using a pool strategy previously tested with saliva samples. In the 2020 study, using the same pool approach in four hospitals (the previous and two additional), and based on 1277 samples, the prevalence was 0.078%.Conclusion: The close temporal coincidence with COVID-19 lockdown suggests that these measures may have had a significant impact on this reduction, although other explanations cannot be ruled-out. What is Known: • Cytomegalovirus is the leading cause of congenital infection. • Behavioural measures decrease cytomegalovirus seroconversion in pregnant women. What is New: • From 2019 to 2020 there was a significant reduction in the prevalence of congenital CMV infection.

Double-blind, Randomized, Placebo-controlled Trial With N-acetylcysteine for Treatment of Severe Acute Respiratory Syndrome Caused by Coronavirus Disease 2019 (COVID-19).

BACKGROUND: A local increase in angiotensin 2 after inactivation of angiotensin-converting enzyme 2 by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) may induce a redox imbalance in alveolar epithelium cells, causing apoptosis, increased inflammation and, consequently, impaired gas exchange. We hypothesized that N-acetylcysteine (NAC) administration could restore this redox homeostasis and suppress unfavorable evolution in patients with coronavirus disease 2019 (COVID-19). METHODS: This was a double-blind, randomized, placebo-controlled, single-center trial conducted at the Emergency Department of Hospital das Clínicas, São Paulo, Brazil, to determine whether NAC in high doses can avoid respiratory failure in patients with COVID-19. We enrolled 135 patients with severe COVID-19 (confirmed or suspected), with an oxyhemoglobin saturation <94% or respiratory rate >24 breaths/minute. Patients were randomized to receive NAC 21 g (~300 mg/kg) for 20 hours or dextrose 5%. The primary endpoint was the need for mechanical ventilation. Secondary endpoints were time of mechanical ventilation, admission to the intensive care unit (ICU), time in ICU, and mortality. RESULTS: Baseline characteristics were similar between the 2 groups, with no significant differences in age, sex, comorbidities, medicines taken, and disease severity. Also, groups were similar in laboratory tests and chest computed tomography scan findings. Sixteen patients (23.9%) in the placebo group received endotracheal intubation and mechanical ventilation, compared with 14 patients (20.6%) in the NAC group (P = .675). No difference was observed in secondary endpoints. CONCLUSIONS: Administration of NAC in high doses did not affect the evolution of severe COVID-19. CLINICAL TRIALS REGISTRATION: Brazilian Registry of Clinical Trials (REBEC): U1111-1250-356 (http://www.ensaiosclinicos.gov.br/rg/RBR-8969zg/).

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Seroprevalence and Risk Factors Among Oligo/Asymptomatic Healthcare Workers: Estimating the Impact of Community Transmission.

We evaluated the seroprevalence of SARS-CoV-2 and risk factors among 4987 oligo/asymptomatic healthcare workers; seroprevalence was 14% and factors associated with SARS-CoV-2 infection were lower educational level (aOR, 1.93; 95% CI, 1.03-3.60), using public transport to work (aOR, 1.65; 95% CI, 1.07-2.62), and working in cleaning or security (aOR, 2.05; 95% CI, 1.04-4.03).

IFCC interim guidelines on rapid point-of-care antigen testing for SARS-CoV-2 detection in asymptomatic and symptomatic individuals.

With an almost unremittent progression of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections all around the world, there is a compelling need to introduce rapid, reliable, and high-throughput testing to allow appropriate clinical management and/or timely isolation of infected individuals. Although nucleic acid amplification testing (NAAT) remains the gold standard for detecting and theoretically quantifying SARS-CoV-2 mRNA in various specimen types, antigen assays may be considered a suitable alternative, under specific circumstances. Rapid antigen tests are meant to detect viral antigen proteins in biological specimens (e.g. nasal, nasopharyngeal, saliva), to indicate current SARS-CoV-2 infection. The available assay methodology includes rapid chromatographic immunoassays, used at the point-of-care, which carries some advantages and drawbacks compared to more conventional, instrumentation-based, laboratory immunoassays. Therefore, this document by the International Federation for Clinical Chemistry and Laboratory Medicine (IFCC) Taskforce on COVID-19 aims to summarize available data on the performance of currently available SARS-CoV-2 antigen rapid detection tests (Ag-RDTs), providing interim guidance on clinical indications and target populations, assay selection, and evaluation, test interpretation and limitations, as well as on pre-analytical considerations. This document is hence mainly aimed to assist laboratory and regulated health professionals in selecting, validating, and implementing regulatory approved Ag-RDTs.

SARS-CoV-2 pneumonia-receptor binding and lung immunopathology: a narrative review.

The current pandemic of COVID-19 caused thousands of deaths and healthcare professionals struggle to properly manage infected patients. This review summarizes information about SARS-CoV-2 receptor binding dynamics and intricacies, lung autopsy findings, immune response patterns, evidence-based explanations for the immune response, and COVID-19-associated hypercoagulability.

Saliva pools for screening of human cytomegalovirus using real-time PCR.

Human cytomegalovirus (HCMV) is the leading congenital infection agent in the world. The importance of screening this infection has been debated, as 10-15% of the asymptomatic newborns with HCMV at birth will present late sequelae. The aim of this study was to test the feasibility of using saliva pools from newborns in a screening program for congenital HCMV infection, in two Portuguese hospitals. The screening was based on the use of pools of 10 saliva samples for detection of viral DNA by real-time PCR. Whenever there was a positive pool, the samples were tested individually, and for each positive sample the result was confirmed with a urine sample collected in the first 2 weeks of life. The study involved 1492 newborns. One hundred and fifty pools were screened, with 14 positive results in saliva, but only 10 were confirmed in urine samples, giving a prevalence of congenital HCMV infection in both hospitals of 0.67% (CI95% 0.36 to 1.23%).Conclusion: The overall prevalence of congenital HCMV infection in both hospitals was 0.67%. The use of saliva pools proved to be effective for the screening of this congenital infection, allowing timely screening and confirmation in a large population, with associated cost reduction. What is Known: • Newborn screening for HCMV is desirable. • Saliva is a good and practical sample. What is New: • The feasibility of using saliva pools for a large-scale screening. • The cost reduction of this strategy.

IFCC Interim Guidelines on Molecular Testing of SARS-CoV-2 Infection.

The diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection globally has relied extensively on molecular testing, contributing vitally to case identification, isolation, contact tracing, and rationalization of infection control measures during the coronavirus disease 2019 (COVID-19) pandemic. Clinical laboratories have thus needed to verify newly developed molecular tests and increase testing capacity at an unprecedented rate. As the COVID-19 pandemic continues to pose a global health threat, laboratories continue to encounter challenges in the selection, verification, and interpretation of these tests. This document by the International Federation for Clinical Chemistry and Laboratory Medicine (IFCC) Task Force on COVID-19 provides interim guidance on: (A) clinical indications and target populations, (B) assay selection, (C) assay verification, and (D) test interpretation and limitations for molecular testing of SARS-CoV-2 infection. These evidence-based recommendations will provide practical guidance to clinical laboratories worldwide and highlight the continued importance of laboratory medicine in our collective pandemic response.

IFCC Interim Guidelines on Serological Testing of Antibodies against SARS-CoV-2.

Serological testing for the detection of antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is emerging as an important component of the clinical management of patients with coronavirus disease 2019 (COVID-19) as well as the epidemiological assessment of SARS-CoV-2 exposure worldwide. In addition to molecular testing for the detection of SARS-CoV-2 infection, clinical laboratories have also needed to increase testing capacity to include serological evaluation of patients with suspected or known COVID-19. While regulatory approved serological immunoassays are now widely available from diagnostic manufacturers globally, there is significant debate regarding the clinical utility of these tests, as well as their clinical and analytical performance requirements prior to application. This document by the International Federation for Clinical Chemistry and Laboratory Medicine (IFCC) Taskforce on COVID-19 provides interim guidance on: (A) clinical indications and target populations, (B) assay selection, (C) assay evaluation, and (D) test interpretation and limitations for serological testing of antibodies against SARS-CoV-2 infection. These evidence-based recommendations will provide practical guidance to clinical laboratories in the selection, verification, and implementation of serological assays and are of the utmost importance as we expand our pandemic response from initial case tracing and containment to mitigation strategies to minimize resurgence and further morbidity and mortality.

IFCC Interim Guidelines on Biochemical/Hematological Monitoring of COVID-19 Patients.

Routine biochemical and hematological tests have been reported to be useful in the stratification and prognostication of pediatric and adult patients with diagnosed coronavirus disease (COVID-19), correlating with poor outcomes such as the need for mechanical ventilation or intensive care, progression to multisystem organ failure, and/or death. While these tests are already well established in most clinical laboratories, there is still debate regarding their clinical value in the management of COVID-19, particularly in pediatrics, as well as the value of composite clinical risk scores in COVID-19 prognostication. This document by the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) Task Force on COVID-19 provides interim guidance on: (A) clinical indications for testing, (B) recommendations for test selection and interpretation, (C) considerations in test interpretation, and (D) current limitations of biochemical/hematological monitoring of COVID-19 patients. These evidence-based recommendations will provide practical guidance to clinical laboratories worldwide, underscoring the contribution of biochemical and hematological testing to our collective pandemic response.

A rare diarrheic parasite in a kidney transplant patient: Cystoisospora belli.

Cystoisospora belli colitis is a rare complication of immunosuppression in solid organ transplant recipients. We describe a case of Cystoisospora belli infection with colitis following renal transplantation.

Monitoring the Efficacy of Chloroquine-Primaquine Therapy for Uncomplicated Plasmodium vivax Malaria in the Main Transmission Hot Spot of Brazil.

Emerging Plasmodium vivax resistance to chloroquine (CQ) may undermine malaria elimination efforts in South America. CQ-resistant P. vivax has been found in the major port city of Manaus but not in the main malaria hot spots across the Amazon Basin of Brazil, where CQ is routinely coadministered with primaquine (PQ) for radical cure of vivax malaria. Here we randomly assigned 204 uncomplicated vivax malaria patients from Juruá Valley, northwestern Brazil, to receive either sequential (arm 1) or concomitant (arm 2) CQ-PQ treatment. Because PQ may synergize the blood schizontocidal effect of CQ and mask low-level CQ resistance, we monitored CQ-only efficacy in arm 1 subjects, who had PQ administered only at the end of the 28-day follow-up. We found adequate clinical and parasitological responses in all subjects assigned to arm 2. However, 2.2% of arm 1 patients had microscopy-detected parasite recrudescences at day 28. When PCR-detected parasitemias at day 28 were considered, response rates decreased to 92.1% and 98.8% in arms 1 and 2, respectively. Therapeutic CQ levels were documented in 6 of 8 recurrences, consistent with true CQ resistance in vivo In contrast, ex vivo assays provided no evidence of CQ resistance in 49 local P. vivax isolates analyzed. CQ-PQ coadministration was not found to potentiate the antirelapse efficacy of PQ over 180 days of surveillance; however, we suggest that larger studies are needed to examine whether and how CQ-PQ interactions, e.g., CQ-mediated inhibition of PQ metabolism, modulate radical cure efficacy in different P. vivax-infected populations. (This study has been registered at ClinicalTrials.gov under identifier NCT02691910.).

P2X7 receptor drives Th1 cell differentiation and controls the follicular helper T cell population to protect against Plasmodium chabaudi malaria.

A complete understanding of the mechanisms underlying the acquisition of protective immunity is crucial to improve vaccine strategies to eradicate malaria. However, it is still unclear whether recognition of damage signals influences the immune response to Plasmodium infection. Adenosine triphosphate (ATP) accumulates in infected erythrocytes and is released into the extracellular milieu through ion channels in the erythrocyte membrane or upon erythrocyte rupture. The P2X7 receptor senses extracellular ATP and induces CD4 T cell activation and death. Here we show that P2X7 receptor promotes T helper 1 (Th1) cell differentiation to the detriment of follicular T helper (Tfh) cells during blood-stage Plasmodium chabaudi malaria. The P2X7 receptor was activated in CD4 T cells following the rupture of infected erythrocytes and these cells became highly responsive to ATP during acute infection. Moreover, mice lacking the P2X7 receptor had increased susceptibility to infection, which correlated with impaired Th1 cell differentiation. Accordingly, IL-2 and IFNγ secretion, as well as T-bet expression, critically depended on P2X7 signaling in CD4 T cells. Additionally, P2X7 receptor controlled the splenic Tfh cell population in infected mice by promoting apoptotic-like cell death. Finally, the P2X7 receptor was required to generate a balanced Th1/Tfh cell population with an improved ability to transfer parasite protection to CD4-deficient mice. This study provides a new insight into malaria immunology by showing the importance of P2X7 receptor in controlling the fine-tuning between Th1 and Tfh cell differentiation during P. chabaudi infection and thus in disease outcome.

Enhanced Ex Vivo Plasmodium vivax Intraerythrocytic Enrichment and Maturation for Rapid and Sensitive Parasite Growth Assays.

Plasmodium vivax chloroquine resistance has been documented in nearly every region where this malaria-causing parasite is endemic. Unfortunately, P. vivax resistance surveillance and drug discovery are challenging due to the low parasitemias of patient isolates and poor parasite survival through ex vivo maturation that reduce the sensitivity and scalability of current P. vivax antimalarial assays. Using cryopreserved patient isolates from Brazil and fresh patient isolates from India, we established a robust enrichment method for P. vivax parasites. We next performed a medium screen for formulations that enhance ex vivo survival. Finally, we optimized an isotopic metabolic labeling assay for measuring P. vivax maturation and its sensitivity to antimalarials. A KCl Percoll density gradient enrichment method increased parasitemias from small-volume ex vivo isolates by an average of >40-fold. The use of Iscove's modified Dulbecco's medium for P. vivax ex vivo culture approximately doubled the parasite survival through maturation. Coupling these with [3H]hypoxanthine metabolic labeling permitted sensitive and robust measurements of parasite maturation, which was used to measure the sensitivities of Brazilian P. vivax isolates to chloroquine and several novel antimalarials. These techniques can be applied to rapidly and robustly assess the P. vivax isolate sensitivities to antimalarials for resistance surveillance and drug discovery.