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ObjectiveTo externally validate various prognostic models and scoring rules for predicting short term mortality in patients admitted to hospital for covid-19.DesignTwo stage individual participant data meta-analysis.SettingSecondary and tertiary care.Participants46 914 patients across 18 countries, admitted to a hospital with polymerase chain reaction confirmed covid-19 from November 2019 to April 2021.Data sourcesMultiple (clustered) cohorts in Brazil, Belgium, China, Czech Republic, Egypt, France, Iran, Israel, Italy, Mexico, Netherlands, Portugal, Russia, Saudi Arabia, Spain, Sweden, United Kingdom, and United States previously identified by a living systematic review of covid-19 prediction models published inThe BMJ, and through PROSPERO, reference checking, and expert knowledge.Model selection and eligibility criteriaPrognostic models identified by the living systematic review and through contacting experts. A priori models were excluded that had a high risk of bias in the participant domain of PROBAST (prediction model study risk of bias assessment tool) or for which the applicability was deemed poor.MethodsEight prognostic models with diverse predictors were identified and validated. A two stage individual participant data meta-analysis was performed of the estimated model concordance (C) statistic, calibration slope, calibration-in-the-large, and observed to expected ratio (O:E) across the included clusters.Main outcome measures30 day mortality or in-hospital mortality.ResultsDatasets included 27 clusters from 18 different countries and contained data on 46 914patients. The pooled estimates ranged from 0.67 to 0.80 (C statistic), 0.22 to 1.22 (calibration slope), and 0.18 to 2.59 (O:E ratio) and were prone to substantial between study heterogeneity. The 4C Mortality Score by Knight et al (pooled C statistic 0.80, 95% confidence interval 0.75 to 0.84, 95% prediction interval 0.72 to 0.86) and clinical model by Wang et al (0.77, 0.73 to 0.80, 0.63 to 0.87) had the highest discriminative ability. On average, 29% fewer deaths were observed than predicted by the 4C Mortality Score (pooled O:E 0.71, 95% confidence interval 0.45 to 1.11, 95% prediction interval 0.21 to 2.39), 35% fewer than predicted by the Wang clinical model (0.65, 0.52 to 0.82, 0.23 to 1.89), and 4% fewer than predicted by Xie et al’s model (0.96, 0.59 to 1.55, 0.21 to 4.28).ConclusionThe prognostic value of the included models varied greatly between the data sources. Although the Knight 4C Mortality Score and Wang clinical model appeared most promising, recalibration (intercept and slope updates) is needed before implementation in routine care.

Background: Recently developed blood tubes with a barrier to provide plasma are becoming widespread. We compared 43 biochemical, 35 immunochemical and 7 serology analytes in a BD-Vacutainer® Barricor tube for local clinical validation of this lithium-heparin tube with a barrier. Methods: Samples from 70 volunteers were collected in different BD-tubes: a clot-activator tube with gel (SST), a lithium-heparin tube with gel (PST), and a lithium-heparin tube with barrier (BAR). Biases from Bland-Altman plots and 95% confidence intervals were compared with the desirable specification from the Ricos database in order to verify whether measurements from different tubes were significantly different. Results: For most of the analytes tested, the measurements using SST, PST or BAR tubes were equivalent. Only BIC, GLU, K, LAD, LPA, P, TP, CTX, Ferritin, HGH, vitD3 and ANTIS showed statistically significant, between-tubes, differences which might have clinical implication. Conclusions: The study demonstrates that SST, PST and BAR can be used interchangeably for most of the analytes tested, including serology analytes. This allows the use of the same tube for assaying multiple analytes, increasing the laboratory efficiency while decreasing patients discomfort by minimizing blood withdrawal. (www.actabiomedica.it)

Objectives The rRT-PCR test, the current gold standard for the detection of coronavirus disease (COVID-19), presents with known shortcomings, such as long turnaround time, potential shortage of reagents, false-negative rates around 15–20%, and expensive equipment. The hematochemical values of routine blood exams could represent a faster and less expensive alternative. Methods Three different training data set of hematochemical values from 1,624 patients (52% COVID-19 positive), admitted at San Raphael Hospital (OSR) from February to May 2020, were used for developing machine learning (ML) models: the complete OSR dataset (72 features: complete blood count (CBC), biochemical, coagulation, hemogasanalysis and CO-Oxymetry values, age, sex and specific symptoms at triage) and two sub-datasets (COVID-specific and CBC dataset, 32 and 21 features respectively). 58 cases (50% COVID-19 positive) from another hospital, and 54 negative patients collected in 2018 at OSR, were used for internal-external and external validation. Results We developed five ML models: for the complete OSR dataset, the area under the receiver operating characteristic curve (AUC) for the algorithms ranged from 0.83 to 0.90; for the COVID-specific dataset from 0.83 to 0.87; and for the CBC dataset from 0.74 to 0.86. The validations also achieved good results: respectively, AUC from 0.75 to 0.78; and specificity from 0.92 to 0.96. Conclusions ML can be applied to blood tests as both an adjunct and alternative method to rRT-PCR for the fast and cost-effective identification of COVID-19-positive patients. This is especially useful in developing countries, or in countries facing an increase in contagions.

P2X7 is a membrane receptor for extracellular ATP which is highly expressed in dendritic cells, macrophages and microglia where it mediates pro-inflammatory responses. The antibiotic polymyxin B, which binds to and neutralizes the toxic residue of bacterial lipopolysaccharide, greatly amplifies cellular responses mediated by the P2X7 receptor. However, the molecular mechanism involved is so far unknown.We investigated the effects of polymyxin B and polymyxin B nonapeptide (PMBN) which is the deacylated amino derivative of polymyxin B lacking the N-terminal fatty amino acid 6-methylheptanoic/octanoic-Dab residue, in human macrophages and HEK293 cells stably expressing the human P2X7 receptor (HEK293-hP2X7). Differences between the two antibiotics were assessed by monitoring the following: nucleotide-induced cytoplasmic free Ca2+ concentration changes, plasma membrane permeability changes, lactate dehydrogenase activity, cell morphology changes. Western blot and microscopic analyses of P2X7GFP-expressing cells were also performed.In contrast to polymyxin B, the polymyxin B nonapeptide was unable to potentiate: a) the ATP-induced Ca2+ increase, b) pore formation and consequently ATP-mediated plasma membrane permeabilization; c) ATP-dependent cytotoxicity. Moreover, in contrast to polymyxin B, polymyxin B nonapeptide did not affect aggregation of the P2X7 receptor subunits and it did not potentiate P2X7-dependent cell fusion.The effects of polymyxin B depended on the presence of its N-terminal fatty amino acid 6-methylheptanoic/octanoic-Dab residue as deletion of this residue abolished polymyxin B-dependent modulation of ATP-triggered responses. These findings are important in the search for allosteric modulators of the P2X7 receptor.

1. Extracellular nucleotides are the focus of increasing attention for their role as extracellular mediators since they are released into the extracellular environment in a regulated manner and/or as a consequence of cell damage. 2. Here, we show that human eosinophils stimulated with different nucleotides release eosinophil cationic protein (ECP) and the chemokine interleukin 8 (IL-8), and that release of these two proteins has a different nucleotide requirement. 3. Release of ECP was triggered in a dose-dependent manner by ATP, UTP and UDP, but not by 2'-3'-o-(4-benzoyl-benzoyl)adenosine 5'-triphosphate (BzATP), ADP and alpha,beta-methylene adenosine 5' triphosphate (alpha,beta-meATP). Release of IL-8 was triggered by UDP, ATP, alpha,beta-meATP and BzATP, but not by UTP or ADP. Pretreatment with pertussis toxin abrogated nucleotide-stimulated ECP but not IL-8 release. 4. Release of IL-8 stimulated by BzATP was fully blocked by the P2X(7) blocker KN-62, while release triggered by ATP was only partially inhibited. IL-8 secretion due to UDP was fully insensitive to KN-62 inhibition. 5. Priming of eosinophils with GM-CSF increased IL-8 secretion irrespectively of the nucleotide used as a stimulant. 6. It is concluded that extracellular nucleotides trigger secretion of ECP by stimulating a receptor of the P2Y subfamily (possibly P2Y(2)), while, on the contrary, nucleotide-stimulated secretion of IL-8 can be due to activation of both P2Y (P2Y(6)) and P2X (P2X(1) and P2X(7)) receptors.

(1) Background: Our aim is the evaluation of the neutralizing activity of BNT162b2 mRNA vaccine-induced antibodies in different in vitro cellular models, as this still represents one of the surrogates of protection against SARS-CoV-2 viral variants. (2) Methods: The entry mechanisms of SARS-CoV-2 in three cell lines (Vero E6, Vero E6/TMPRSS2 and Calu-3) were evaluated with both pseudoviruses and whole virus particles. The neutralizing capability of sera collected from vaccinated subjects was characterized through cytopathic effects and Real-Time RT PCR. (3) Results: In contrast to Vero E6 and Vero E6/TMPRSS2, Calu-3 allowed the evaluation of both viral entry mechanisms, resembling what occurs during natural infection. The choice of an appropriate cellular model can decisively influence the determination of the neutralizing activity of antibodies against SARS-CoV-2 variants. Indeed, the lack of correlation between neutralizing data in Calu-3 and Vero E6 demonstrated that testing the antibody inhibitory activity by using a single cell model possibly results in an inaccurate characterization. (4) Conclusions: Cellular systems allowing only one of the two viral entry pathways may not fully reflect the neutralizing activity of vaccine-induced antibodies moving increasingly further away from possible correlates of protection from SARS-CoV-2 infection.

Background Serological tests can be used to detect antibodies in the serum of subject's after SARS-CoV-2 infection and vaccination. Currently, variability in antibody titers and the availability of a multiplicity of serological tests have made it necessary to highlight their appropriateness and limitations in various diagnostic settings. Methods This study is part of Covidiagnostix, a multicenter project aimed at the assessment of the health technology used in SARS-CoV-2 serological tests. Based on data gained from the analysis of over 5000 subjects, a selected number of serum samples, representative of different diagnostic settings, were analyzed first by qualitative immunoassays (IgA, M, and G MILLIPLEX(R) SARS-CoV-2 tests based on Luminex(R)) to define the immunoglobulins serum composition and subsequently by four serological diagnostic tests (Elecsys Anti-SARS-CoV-2 and Elecsys Anti-SARS-CoV-2 S by Roche, SARS-CoV-2 IgG by Siemens Healthcare, and CHORUS SARS-CoV-2 "NEUTRALIZING" Ab by DIESSE). The first WHO International Standard for SARS-CoV-2 was also analyzed using the same methods. Results This study evaluated the antibody content and titer of the WHO Standard and serum of subjects with/without previous infection and before/after vaccination for SARS-CoV-2. Conclusion The definition of antibodies in the WHO standard and the analysis of serum samples allowed for the identification of the appropriateness of serological tests in each diagnostic setting, increasing the effectiveness of the resulting laboratory data. Furthermore, we found that it would be optimal to produce new international standards against the S1 domain and RBD of the SARS-CoV-2 spike protein for a more effective serological monitoring of vaccination.

Purpose: To compare sleeve gastrectomy (SG) to SG associated with Rossetti fundoplication (SG + RF) in terms of de novo gastro-esophageal reflux disease (GERD) after surgery, weight loss, and postoperative complications. Materials and methods: Patients affected by morbid obesity, without symptoms of GERD, who were never in therapy with proton pump inhibitors (PPIs), were randomized into two groups. One group underwent SG and the other SG + RF. The study was stopped on February 2020 due to the COVID pandemic. Results: A total of 278 patients of the programmed number of 404 patients were enrolled (68.8%). De novo esophagitis was considered in those patients who had both pre- and postoperative gastroscopy (97/278, 34.9%). Two hundred fifty-one patients (90.3%) had completed clinical follow-up at 12months. SG + RF resulted in an adequate weight loss, similar to classic SG at 12-month follow-up (%TWL = 35. 4 ± 7.2%) with a significantly better outcome in terms of GERD development. One year after surgery, PPIs were necessary in 4.3% SG + RF patients compared to 17.1% SG patients (p = 0.001). Esophagitis was present in 2.0% of SG + RF patients versus 23.4% SG patients (p = 0.002). The main complication after SG + RF was wrap perforation (4.3%), which improved with the surgeon's learning curve. Conclusion: SG + RF seemed to be an effective alternative to classic SG in preventing de novo GERD. More studies are needed to establish that an adequate learning curve decreases the higher percentage of short-term complications in the SG + RF group.

Background Real-world population studies have shown waning immunity, over time, after receiving the two doses of the BNT162b2 COVID-19 vaccine. Studies reporting the long-term humoral response are important to drive future vaccination strategies like the introduction of the booster dose. Yet, available literature on long follow-up periods is scarce. Covidiagnostix is a multicenter study aiming to assess the antibody response in >1000 healthcare professionals (HCPs) who received the BNT162b2 vaccine. Methods Serum was tested at time-0 (T0), before the first dose and then at T1, T2, T3 and T4, respectively, 21, 42, 177 and 302days after T0. Antibodies against the SARS-CoV-2 nucleocapsid-protein were measured to assess SARS-CoV-2 infections, whereas antibodies against the receptor-binding domain of the spike protein were measured to assess vaccine response. Results The antibody titer observed 10months post-vaccination showed a decrease of approximately 80% from the peak measured at T2, yet the median titer of the seronegatives HCPs was still higher than seropositives before vaccination. We identified 12 post-vaccination infected HCPs within 6months after receiving the first dose and another 12 post-vaccination infected HCPs between 6 and 10months post-vaccination. Conclusion Vaccination induced a humoral response which is well detectable even 10months post-vaccination. Yet a high anti-spike serum antibody titer does not guarantee protection from infection. Differences in symptomatology between SARS-CoV-2 infections occurred within the first 6months post-vaccination and the following 4months, and differences in COVID-19 prevalence and vaccination coverage observed in these two time intervals were consistent with a decrease in vaccine efficacy 6months after receiving the first dose.

SARS-CoV-2 spike is evolving to maximize transmissibility and evade the humoral response. The massive genomic sequencing of SARS-CoV-2 isolates has led to the identification of single-point mutations and deletions, often having the recurrence of hotspots, associated with advantageous phenotypes. We report the isolation and molecular characterization of a SARS-CoV-2 strain, belonging to a lineage (C.36) not previously associated with concerning traits, which shows decreased susceptibility to vaccine sera neutralization.

Vincenzo Spagnuolo,1,2 Laura Galli,2 Andrea Poli,2 Alba Bigoloni,2 Luca Fumagalli,2 Nicola Gianotti,2 Silvia Nozza,2 Davide Ferrari,3 Massimo Locatelli,3 Adriano Lazzarin,2 Antonella Castagna1,2 1Vita-Salute San Raffaele University, Faculty of Medicine and Surgery, Milan, Italy; 2Clinic of Infectious Diseases, Istituto di Ricerca e Cura a Carattere Scientifico (IRCCS) San Raffaele Scientific Institute, Milan, Italy; 3Laboratory Medicine Service, IRCCS San Raffaele Hospital, Milan, Italy IntroductionThe increased exposure of dolutegravir (DTG) when given with atazanavir/ritonavir(ATV/r), as well as the acceptable safety profile, may suggest the use of this combinationas a two-drug regimen both in virologically suppressed and treatment-failingsubjects.1–5 This nucleoside reverse transcriptase inhibitors (NRTIs)-sparing regimen, characterizedby a high genetic barrier, may represent an option in patients who have failedprevious regimens and developed pharmacoresistance mutations to NRTIs and nonnucleosidereverse transcriptase inhibitors (NNRTIs). However, no data on DTG plus boosted ATV in patients with virological failureare currently available. Therefore, the aim of the DOLATAV study was to investigate the efficacy, safety,and pharmacokinetics of ATV/r 300/100 mg once-daily plus DTG 50 mg once-daily &nbsp

Background The pilot Phase IIb VIKING study suggested that dolutegravir (DTG), an HIV integrase inhibitor (INI), is efficacious in INI-resistant patients at the 50 mg twice-daily dose. However, DTG response was most reduced in subjects carrying resistance-associated mutations at position G140 and Q148. These mutations can cause a 10–20-fold reduced susceptibility to DTG as well as a 96% lower odds of achieving HIV-1 RNA Methods Five multi-experienced patients harbouring the mutation complex G140-Q148, resistant to at least three drug classes, and previously exposed to DTG 50 mg twice daily, were treated with an increased dose of DTG (100 mg twice daily) in association with an optimized background regimen (OBR) based on their individual viral genotyping assays. The blood concentration of DTG was measured in order to determine whether a solubility issue is related with this high dosage. Results Four out of five patients attained an HIV-1 RNA Conclusions For the first time 100 mg twice daily of DTG was administered to five multi-experienced patients harbouring the mutation complex G140-Q148. Although a small number of patients were tested, the results show a potential for a high-dose regimen of DTG as a rescue therapy in patients harbouring integrase strand transfer inhibitor resistant viruses.

Antibiotic therapy is a crucial aspect of the management of hospitalized patients, however, current standard dosing protocols have been shown to often attain inadequate plasmatic concentrations which may impair the clinical outcome and promote the selection of multidrug-resistant bacteria. The aim of this study is to establish and validate a robust and fast liquid chromatography-tandem mass spectrometry (LC–MS/MS) method for the simultaneous analysis of four commonly used antibiotics (Meropenem, Piperacillin, Linezolid and Teicoplanin) in human plasma according to the European Medicines Agency (EMA) guidelines. Samples preparation was performed using a commercially available extraction kit which needs a very small amount of sample (50 μl). Antibiotics were detected, following a 7 min gradient separation, in multiple reactions monitoring (MRM) mode using a Qtrap 5500 triple quadrupole instrument equipped with an electrospray source operating in positive ion mode. The method, covering the antibiotics’ clinically relevant concentration ranges, is also able to quantify, individually, the major teicoplanin components. The high reproducibility and the need of a small amount of sample, associated with the use of a commercial kit, together with a short chromatographic time, makes the method particularly suited for high-throughput routine analysis. Monitoring of plasma antibiotic levels, as part of the clinical routine, would result in a quick therapy adjustment leading to a higher probability of eradicating the infection as well as a potential reduction of multidrug-resistance prevalence. The method was successfully applied to monitor the antibiotic concentration of 49 patients under therapy.

Driving under the influence of illicit drugs (DUID) represents a significant menace to public safety and is therefore sanctioned with severe fines and penalties such as driving disqualification or even arrest in case the accident has caused serious injury or death. In Italy, DUID is regulated by the article 187 of the National Street Code, however, the list of the substances to be searched and their threshold concentrations are left to the 20 Italian regional authorities. A further lack of legislative standardization concerns the type of detection methods and moreover the time gap between the car accident and blood sampling. This interval can be as high as 5 h, enough to significantly reduce the concentration of drugs with fast pharmacokinetic. By analyzing 1258 blood tests performed on drivers involved in road traffic crashes in the Milan area between 2012 and 2016 we show that approximately 75% of such drivers who tested positive for THC and 15% of the drivers who tested positive for cocaine are at risk of misjudgment. Considering the severe sanctions associated with DUID, we emphasize the urgency of introducing a corrective factor that takes into account the time elapsed between the accident and blood sampling in order to avoid unfair treatment, including the unjust application of sanctions.

Background Driving under the influence of alcohol and/or illicit drugs in Italy is regulated by the articles 186 and 187 of the National Street Code. Epidemiological studies on drivers involved in road traffic crashes (RTC) provide useful information about the use/abuse of these substances in the general population. Comparison with case control studies may reveal important information like the cut-off limits adequacy. Methods Data from 1587 blood tests for alcohol and 1258 blood tests for illicit drugs on drivers involved in RTC around Milan between 2012 and 2016, were analyzed and compared with a published random survey (DRUID) from the European Community. Results Our data from RTC-involved drivers show that alcohol abuse is not age-related whereas illicit drugs are more common in young people. Cannabinoids are frequent among younger drivers (median age 27) whereas cocaine is more often detected in adults (median age 34). The calculated odds ratio after comparison with the DRUID survey shows that a blood alcohol concentration below the legal limit does not represent a risk factor in having a car accident whereas concentrations of cocaine and cannabinoids within the legal limits are associated with being involved in a car accident. Conclusions Despite authority efforts, the abuse of alcohol and illicit drugs is still common in young drivers. We suspect that the cut-off limits for cannabinoids and cocaine and/or the pre-analytical procedures for these substances are inadequate. We suggest a better standardization of the procedure by shortening the time interval between the request for investigation and blood collection and propose the adoption of more stringent cut-off limits.

Unlike other vitamins, the vitamin D concentration in blood varies cyclically over the course of the year in relation to genetic (gender, ethnicity, polymorphisms) and environmental factors (sunlight exposure, diet, food-related or direct vitamin D supplementation, skin pigmentation). Although the major diagnostics manufacturers have recently developed improved automated 25-hydroxy vitamin D immunoassays, the intra- and inter-laboratory variability is still high (especially at low vitamin D concentrations) which might lead to incorrect vitamin D deficiency/insufficiency diagnosis. Moreover, despite recent efforts to standardize the assay and minimize its variability, the current bias for measured vitamin D concentrations is often still above the desirable ± 10% criterion. Because the implications of low vitamin D concentrations in non-skeletal diseases are still partially unknown, international guideline recommendations for establishing meaningful ranges, at any time over the course of the year, irrespective not only of environmental and personal factors but also of instrumental variability, are needed. In this review, we discuss the main factors that influence the variability of vitamin D concentrations and whether a centile curve, individually calculated by a theoretical equation considering such factors, might be better suited than a fixed limit to assess abnormal vitamin D concentrations in otherwise healthy subjects. Vitamin D reference ranges during pregnancy, childhood, or diagnosed illnesses, which merit separate discussion, are beyond the scope of this review.

Adenosine (ADO) and nucleotides such as ATP, ADP, and uridine 5′-triphosphate (UTP), among others, may serve as extracellular signaling molecules. These mediators activate specific cell-surface receptors—namely, purinergic 1 and 2 (P1 and P2)—to modulate crucial pathophysiological responses. Regulation of this process is maintained by nucleoside and nucleotide transporters, as well as the ectonucleotidases ectonucleoside triphosphate diphosphohydrolase [ENTPD; cluster of differentiation (CD)39] and ecto-5′-nucleotidase (5′-NT; CD73), among others. Cells involved in tissue repair, healing, and scarring respond to both ADO and ATP. Our recent investigations have shown that modulation of purinergic signaling regulates matrix deposition during tissue repair and fibrosis in several organs. Cells release adenine nucleotides into the extracellular space, where these mediators are converted by CD39 and CD73 into ADO, which is anti-inflammatory in the short term but may also promote dermal, heart, liver, and lung fibrosis with repetitive signaling under defined circumstances. Extracellular ATP stimulates cardiac fibroblast proliferation, lung inflammation, and fibrosis. P2Y2 (UTP/ATP) and P2Y6 [ADP/UTP/uridine 5′-diphosphate (UDP)] have been shown to have profibrotic effects, as well. Modulation of purinergic signaling represents a novel approach to preventing or diminishing fibrosis. We provide an overview of the current understanding of purinergic signaling in scarring and discuss its potential to prevent or decrease fibrosis.—Ferrari, D., Gambari, R., Idzko, M., Müller, T., Albanesi, C., Pastore, S., La Manna, G., Robson, S. C., Cronstein, B. Purinergic signaling in scarring.