Sensitive protein detection using site-specifically oligonucleotide-conjugated nanobody reagents

High-quality affinity probes are critical for sensitive and specific protein detection, in particular for detection of protein biomarkers in the early phases of disease development. Proximity extension assays (PEAs) have been used for high-throughput multiplexed protein detection of up to a few thousand different proteins in one or a few microliters of plasma. Clonal affinity reagents can offer advantages over the commonly used polyclonal antibodies (pAbs) in terms of reproducibility and standardization of such assays. Here, we explore nanobodies (Nbs) as an alternative to pAbs as affinity reagents for PEA. We describe an efficient site-specific approach for preparing high-quality oligo-conjugated Nb probes via enzyme coupling using Sortase A (SrtA). The procedure allows convenient removal of unconjugated affinity reagents after conjugation. The purified high-grade Nb probes were used in PEA, and the reactions provided an efficient means to select optimal pairs of binding reagents from a group of affinity reagents. We demonstrate that Nb-based PEA (nano-PEA) for interleukin-6 (IL6) detection can augment assay performance, compared to the use of pAb probes. We identify and validate Nb combinations capable of binding in pairs without competition for IL6 antigen detection by PEA.We thank Dr. Junhong Yan for discussions about Nbs oligo conjugation. The Single Cell Proteomic Facility at SciLifeLab, Uppsala for supports to setup PEA assay. This work was supported by a grant (2012-5852) from the Swedish Research Council’s special call for future therapy, Swedish Research Council under grant agreement No. 2020-02258, a Torsten Söderbergs Stiftelse (M130/16) and the European Research Council under the European Union's Seventh Framework Programme (FP/2007-2013)/ ERC Grant Agreement No. 294409, ProteinSeq and from The Swedish Collegium for Advanced Studies (SCAS).Alternative recombinant protein binders selection via proximity assay

Geoeconomic variations in epidemiology, ventilation management, and outcomes in invasively ventilated intensive care unit patients without acute respiratory distress syndrome: a pooled analysis of four observational studies

Background: Geoeconomic variations in epidemiology, the practice of ventilation, and outcome in invasively ventilated intensive care unit (ICU) patients without acute respiratory distress syndrome (ARDS) remain unexplored. In this analysis we aim to address these gaps using individual patient data of four large observational studies. Methods: In this pooled analysis we harmonised individual patient data from the ERICC, LUNG SAFE, PRoVENT, and PRoVENT-iMiC prospective observational studies, which were conducted from June, 2011, to December, 2018, in 534 ICUs in 54 countries. We used the 2016 World Bank classification to define two geoeconomic regions: middle-income countries (MICs) and high-income countries (HICs). ARDS was defined according to the Berlin criteria. Descriptive statistics were used to compare patients in MICs versus HICs. The primary outcome was the use of low tidal volume ventilation (LTVV) for the first 3 days of mechanical ventilation. Secondary outcomes were key ventilation parameters (tidal volume size, positive end-expiratory pressure, fraction of inspired oxygen, peak pressure, plateau pressure, driving pressure, and respiratory rate), patient characteristics, the risk for and actual development of acute respiratory distress syndrome after the first day of ventilation, duration of ventilation, ICU length of stay, and ICU mortality. Findings: Of the 7608 patients included in the original studies, this analysis included 3852 patients without ARDS, of whom 2345 were from MICs and 1507 were from HICs. Patients in MICs were younger, shorter and with a slightly lower body-mass index, more often had diabetes and active cancer, but less often chronic obstructive pulmonary disease and heart failure than patients from HICs. Sequential organ failure assessment scores were similar in MICs and HICs. Use of LTVV in MICs and HICs was comparable (42·4% vs 44·2%; absolute difference -1·69 [-9·58 to 6·11] p=0·67; data available in 3174 [82%] of 3852 patients). The median applied positive end expiratory pressure was lower in MICs than in HICs (5 [IQR 5-8] vs 6 [5-8] cm H2O; p=0·0011). ICU mortality was higher in MICs than in HICs (30·5% vs 19·9%; p=0·0004; adjusted effect 16·41% [95% CI 9·52-23·52]; p<0·0001) and was inversely associated with gross domestic product (adjusted odds ratio for a US$10 000 increase per capita 0·80 [95% CI 0·75-0·86]; p<0·0001). Interpretation: Despite similar disease severity and ventilation management, ICU mortality in patients without ARDS is higher in MICs than in HICs, with a strong association with country-level economic status

Ticagrelor in patients with diabetes and stable coronary artery disease with a history of previous percutaneous coronary intervention (THEMIS-PCI) : a phase 3, placebo-controlled, randomised trial

Background: Patients with stable coronary artery disease and diabetes with previous percutaneous coronary intervention (PCI), particularly those with previous stenting, are at high risk of ischaemic events. These patients are generally treated with aspirin. In this trial, we aimed to investigate if these patients would benefit from treatment with aspirin plus ticagrelor. Methods: The Effect of Ticagrelor on Health Outcomes in diabEtes Mellitus patients Intervention Study (THEMIS) was a phase 3 randomised, double-blinded, placebo-controlled trial, done in 1315 sites in 42 countries. Patients were eligible if 50 years or older, with type 2 diabetes, receiving anti-hyperglycaemic drugs for at least 6 months, with stable coronary artery disease, and one of three other mutually non-exclusive criteria: a history of previous PCI or of coronary artery bypass grafting, or documentation of angiographic stenosis of 50% or more in at least one coronary artery. Eligible patients were randomly assigned (1:1) to either ticagrelor or placebo, by use of an interactive voice-response or web-response system. The THEMIS-PCI trial comprised a prespecified subgroup of patients with previous PCI. The primary efficacy outcome was a composite of cardiovascular death, myocardial infarction, or stroke (measured in the intention-to-treat population). Findings: Between Feb 17, 2014, and May 24, 2016, 11 154 patients (58% of the overall THEMIS trial) with a history of previous PCI were enrolled in the THEMIS-PCI trial. Median follow-up was 3·3 years (IQR 2·8–3·8). In the previous PCI group, fewer patients receiving ticagrelor had a primary efficacy outcome event than in the placebo group (404 [7·3%] of 5558 vs 480 [8·6%] of 5596; HR 0·85 [95% CI 0·74–0·97], p=0·013). The same effect was not observed in patients without PCI (p=0·76, p interaction=0·16). The proportion of patients with cardiovascular death was similar in both treatment groups (174 [3·1%] with ticagrelor vs 183 (3·3%) with placebo; HR 0·96 [95% CI 0·78–1·18], p=0·68), as well as all-cause death (282 [5·1%] vs 323 [5·8%]; 0·88 [0·75–1·03], p=0·11). TIMI major bleeding occurred in 111 (2·0%) of 5536 patients receiving ticagrelor and 62 (1·1%) of 5564 patients receiving placebo (HR 2·03 [95% CI 1·48–2·76], p<0·0001), and fatal bleeding in 6 (0·1%) of 5536 patients with ticagrelor and 6 (0·1%) of 5564 with placebo (1·13 [0·36–3·50], p=0·83). Intracranial haemorrhage occurred in 33 (0·6%) and 31 (0·6%) patients (1·21 [0·74–1·97], p=0·45). Ticagrelor improved net clinical benefit: 519/5558 (9·3%) versus 617/5596 (11·0%), HR=0·85, 95% CI 0·75–0·95, p=0·005, in contrast to patients without PCI where it did not, p interaction=0·012. Benefit was present irrespective of time from most recent PCI. Interpretation: In patients with diabetes, stable coronary artery disease, and previous PCI, ticagrelor added to aspirin reduced cardiovascular death, myocardial infarction, and stroke, although with increased major bleeding. In that large, easily identified population, ticagrelor provided a favourable net clinical benefit (more than in patients without history of PCI). This effect shows that long-term therapy with ticagrelor in addition to aspirin should be considered in patients with diabetes and a history of PCI who have tolerated antiplatelet therapy, have high ischaemic risk, and low bleeding risk

Presentation, management, and outcomes of older compared to younger adults with hospital-acquired bloodstream infections in the intensive care unit: a multicenter cohort study

Purpose: Older adults admitted to the intensive care unit (ICU) usually have fair baseline functional capacity, yet their age and frailty may compromise their management. We compared the characteristics and management of older (≥ 75&nbsp;years) versus younger adults hospitalized in ICU with hospital-acquired bloodstream infection (HA-BSI). Methods: Nested cohort study within the EUROBACT-2 database, a multinational prospective cohort study including adults (≥ 18&nbsp;years) hospitalized in the ICU during 2019-2021. We compared older versus younger adults in terms of infection characteristics (clinical signs and symptoms, source, and microbiological data), management (imaging, source control, antimicrobial therapy), and outcomes (28-day mortality and hospital discharge). Results: Among 2111 individuals hospitalized in 219 ICUs with HA-BSI, 563 (27%) were ≥ 75&nbsp;years old. Compared to younger patients, these individuals had higher comorbidity score and lower functional capacity; presented more often with a pulmonary, urinary, or unknown HA-BSI source; and had lower heart rate, blood pressure and temperature at presentation. Pathogens and resistance rates were similar in both groups. Differences in management included mainly lower rates of effective source control achievement among aged individuals. Older adults also had significantly higher day-28 mortality (50% versus 34%, p &lt; 0.001), and lower rates of discharge from hospital (12% versus 20%, p &lt; 0.001) by this time. Conclusions: Older adults with HA-BSI hospitalized in ICU have different baseline characteristics and source of infection compared to younger patients. Management of older adults differs mainly by lower probability to achieve source control. This should be targeted to improve outcomes among older ICU patients

Epidemiology and outcomes of hospital-acquired bloodstream infections in intensive care unit patients: the EUROBACT-2 international cohort study

Purpose In the critically ill, hospital-acquired bloodstream infections (HA-BSI) are associated with significant mortality. Granular data are required for optimizing management, and developing guidelines and clinical trials. Methods We carried out a prospective international cohort study of adult patients (≥ 18 years of age) with HA-BSI treated in intensive care units (ICUs) between June 2019 and February 2021. Results 2600 patients from 333 ICUs in 52 countries were included. 78% HA-BSI were ICU-acquired. Median Sequential Organ Failure Assessment (SOFA) score was 8 [IQR 5; 11] at HA-BSI diagnosis. Most frequent sources of infection included pneumonia (26.7%) and intravascular catheters (26.4%). Most frequent pathogens were Gram-negative bacteria (59.0%), predominantly Klebsiella spp. (27.9%), Acinetobacter spp. (20.3%), Escherichia coli (15.8%), and Pseudomonas spp. (14.3%). Carbapenem resistance was present in 37.8%, 84.6%, 7.4%, and 33.2%, respectively. Difficult-to-treat resistance (DTR) was present in 23.5% and pan-drug resistance in 1.5%. Antimicrobial therapy was deemed adequate within 24 h for 51.5%. Antimicrobial resistance was associated with longer delays to adequate antimicrobial therapy. Source control was needed in 52.5% but not achieved in 18.2%. Mortality was 37.1%, and only 16.1% had been discharged alive from hospital by day-28. Conclusions HA-BSI was frequently caused by Gram-negative, carbapenem-resistant and DTR pathogens. Antimicrobial resistance led to delays in adequate antimicrobial therapy. Mortality was high, and at day-28 only a minority of the patients were discharged alive from the hospital. Prevention of antimicrobial resistance and focusing on adequate antimicrobial therapy and source control are important to optimize patient management and outcomes

The role of centre and country factors on process and outcome indicators in critically ill patients with hospital-acquired bloodstream infections

Purpose: The primary objective of this study was to evaluate the associations between centre/country-based factors and two important process and outcome indicators in patients with hospital-acquired bloodstream infections (HABSI). Methods: We used data on HABSI from the prospective EUROBACT-2 study to evaluate the associations between centre/country factors on a process or an outcome indicator: adequacy of antimicrobial therapy within the first 24&nbsp;h or 28-day mortality, respectively. Mixed logistical models with clustering by centre identified factors associated with both indicators. Results: Two thousand two hundred nine patients from two hundred one intensive care units (ICUs) were included in forty-seven countries. Overall, 51% (n = 1128) of patients received an adequate antimicrobial therapy and the 28-day mortality was 38% (n = 839). The availability of therapeutic drug monitoring (TDM) for aminoglycosides everyday [odds ratio (OR) 1.48, 95% confidence interval (CI) 1.03-2.14] or within a few hours (OR 1.79, 95% CI 1.34-2.38), surveillance cultures for multidrug-resistant organism carriage performed weekly (OR 1.45, 95% CI 1.09-1.93), and increasing Human Development Index (HDI) values were associated with adequate antimicrobial therapy. The presence of intermediate care beds (OR 0.63, 95% CI 0.47-0.84), TDM for aminoglycoside available everyday (OR 0.66, 95% CI 0.44-1.00) or within a few hours (OR 0.51, 95% CI 0.37-0.70), 24/7 consultation of clinical pharmacists (OR 0.67, 95% CI 0.47-0.95), percentage of vancomycin-resistant enterococci (VRE) between 10% and 25% in the ICU (OR 1.67, 95% CI 1.00-2.80), and decreasing HDI values were associated with 28-day mortality. Conclusion: Centre/country factors should be targeted for future interventions to improve management strategies and outcome of HABSI in ICU patients

Respiratory support in patients with severe COVID-19 in the International Severe Acute Respiratory and Emerging Infection (ISARIC) COVID-19 study: a prospective, multinational, observational study

Background: Up to 30% of hospitalised patients with COVID-19 require advanced respiratory support, including high-flow nasal cannulas (HFNC), non-invasive mechanical ventilation (NIV), or invasive mechanical ventilation (IMV). We aimed to describe the clinical characteristics, outcomes and risk factors for failing non-invasive respiratory support in patients treated with severe COVID-19 during the first two years of the pandemic in high-income countries (HICs) and low middle-income countries (LMICs). Methods: This is a multinational, multicentre, prospective cohort study embedded in the ISARIC-WHO COVID-19 Clinical Characterisation Protocol. Patients with laboratory-confirmed SARS-CoV-2 infection who required hospital admission were recruited prospectively. Patients treated with HFNC, NIV, or IMV within the first 24 h of hospital admission were included in this study. Descriptive statistics, random forest, and logistic regression analyses were used to describe clinical characteristics and compare clinical outcomes among patients treated with the different types of advanced respiratory support. Results: A total of 66,565 patients were included in this study. Overall, 82.6% of patients were treated in HIC, and 40.6% were admitted to the hospital during the first pandemic wave. During the first 24 h after hospital admission, patients in HICs were more frequently treated with HFNC (48.0%), followed by NIV (38.6%) and IMV (13.4%). In contrast, patients admitted in lower- and middle-income countries (LMICs) were less frequently treated with HFNC (16.1%) and the majority received IMV (59.1%). The failure rate of non-invasive respiratory support (i.e. HFNC or NIV) was 15.5%, of which 71.2% were from HIC and 28.8% from LMIC. The variables most strongly associated with non-invasive ventilation failure, defined as progression to IMV, were high leukocyte counts at hospital admission (OR [95%CI]; 5.86 [4.83-7.10]), treatment in an LMIC (OR [95%CI]; 2.04 [1.97-2.11]), and tachypnoea at hospital admission (OR [95%CI]; 1.16 [1.14-1.18]). Patients who failed HFNC/NIV had a higher 28-day fatality ratio (OR [95%CI]; 1.27 [1.25-1.30]). Conclusions: In the present international cohort, the most frequently used advanced respiratory support was the HFNC. However, IMV was used more often in LMIC. Higher leucocyte count, tachypnoea, and treatment in LMIC were risk factors for HFNC/NIV failure. HFNC/NIV failure was related to worse clinical outcomes, such as 28-day mortality. Trial registration This is a prospective observational study; therefore, no health care interventions were applied to participants, and trial registration is not applicable