Development and validation of a risk prediction model for hospital admission in COVID-19 patients presenting to primary care

BACKGROUND: There is a paucity of prognostic models for COVID-19 that are usable for in-office patient assessment in general practice (GP). OBJECTIVES: To develop and validate a risk prediction model for hospital admission with readily available predictors. METHODS: A retrospective cohort study linking GP records from 8 COVID-19 centres and 55 general practices in the Netherlands to hospital admission records. The development cohort spanned March to June 2020, the validation cohort March to June 2021. The primary outcome was hospital admission within 14 days. We used geographic leave-region-out cross-validation in the development cohort and temporal validation in the validation cohort. RESULTS: In the development cohort, 4,806 adult patients with COVID-19 consulted their GP (median age 56, 56% female); in the validation cohort 830 patients did (median age 56, 52% female). In the development and validation cohort respectively, 292 (6.1%) and 126 (15.2%) were admitted to the hospital within 14 days, respectively. A logistic regression model based on sex, smoking, symptoms, vital signs and comorbidities predicted hospital admission with a c-index of 0.84 (95% CI 0.83 to 0.86) at geographic cross-validation and 0.79 (95% CI 0.74 to 0.83) at temporal validation, and was reasonably well calibrated (intercept -0.08, 95% CI -0.98 to 0.52, slope 0.89, 95% CI 0.71 to 1.07 at geographic cross-validation and intercept 0.02, 95% CI -0.21 to 0.24, slope 0.82, 95% CI 0.64 to 1.00 at temporal validation). CONCLUSION: We derived a risk model using readily available variables at GP assessment to predict hospital admission for COVID-19. It performed accurately across regions and waves. Further validation on cohorts with acquired immunity and newer SARS-CoV-2 variants is recommended

Implementation of Novel Molecular Biomarkers for Non-small Cell Lung Cancer in the Netherlands:How to Deal With Increasing Complexity

The diagnostic landscape of non-small cell lung cancer (NSCLC) is changing rapidly with the availability of novel treatments. Despite high-level healthcare in the Netherlands, not all patients with NSCLC are tested with the currently relevant predictive tumor markers that are necessary for optimal decision-making for today's available targeted or immunotherapy. An expert workshop on the molecular diagnosis of NSCLC involving pulmonary oncologists, clinical chemists, pathologists, and clinical scientists in molecular pathology was held in the Netherlands on December 10, 2018. The aims of the workshop were to facilitate cross-disciplinary discussions regarding standards of practice, and address recent developments and associated challenges that impact future practice. This paper presents a summary of the discussions and consensus opinions of the workshop participants on the initial challenges of harmonization of the detection and clinical use of predictive markers of NSCLC. A key theme identified was the need for broader and active participation of all stakeholders involved in molecular diagnostic services for NSCLC, including healthcare professionals across all disciplines, the hospitals and clinics involved in service delivery, healthcare insurers, and industry groups involved in diagnostic and treatment innovations. Such collaboration is essential to integrate different technologies into molecular diagnostics practice, to increase nationwide patient access to novel technologies, and to ensure consensus-preferred biomarkers are tested

Dutch National Round Robin Trial on Plasma-Derived Circulating Cell-Free DNA Extraction Methods Routinely Used in Clinical Pathology for Molecular Tumor Profiling

BACKGROUND: Efficient recovery of circulating tumor DNA (ctDNA) depends on the quantity and quality of circulating cell-free DNA (ccfDNA). Here, we evaluated whether various ccfDNA extraction methods routinely applied in Dutch laboratories affect ccfDNA yield, ccfDNA integrity, and mutant ctDNA detection, using identical lung cancer patient-derived plasma samples. METHODS: Aliquots of 4 high-volume diagnostic leukapheresis plasma samples and one artificial reference plasma sample with predetermined tumor-derived mutations were distributed among 14 Dutch laboratories. Extractions of ccfDNA were performed according to local routine standard operating procedures and were analyzed at a central reference laboratory for mutant detection and assessment of ccfDNA quantity and integrity. RESULTS: Mutant molecule levels in extracted ccfDNA samples varied considerably between laboratories, but there was no indication of consistent above or below average performance. Compared to silica membrane-based methods, samples extracted with magnetic beads-based kits revealed an overall lower total ccfDNA yield (-29%; P < 0.0001) and recovered fewer mutant molecules (-41%; P < 0.01). The variant allelic frequency and sample integrity were similar. In samples with a higher-than-average total ccfDNA yield, an augmented recovery of mutant molecules was observed. CONCLUSIONS: In the Netherlands, we encountered diversity in preanalytical workflows with potential consequences on mutant ctDNA detection in clinical practice. Silica membrane-based methodologies resulted in the highest total ccfDNA yield and are therefore preferred to detect low copy numbers of relevant mutations. Harmonization of the extraction workflow for accurate quantification and sensitive detection is required to prevent introduction of technical divergence in the preanalytical phase and reduce interlaboratory discrepancies

A novel haemocytometric COVID-19 prognostic score developed and validated in an observational multicentre European hospital-based study

COVID-19 induces haemocytometric changes. Complete blood count changes, including new cell activation parameters, from 982 confirmed COVID-19 adult patients from 11 European hospitals were retrospectively analysed for distinctive patterns based on age, gender, clinical severity, symptom duration, and hospital days. The observed haemocytometric patterns formed the basis to develop a multi-haemocytometric-parameter prognostic score to predict, during the first three days after presentation, which patients will recover without ventilation or deteriorate within a two-week timeframe, needing intensive care or with fatal outcome. The prognostic score, with ROC curve AUC at baseline of 0.753 (95% CI 0.723-0.781) increasing to 0.875 (95% CI 0.806-0.926) on day 3, was superior to any individual parameter at distinguishing between clinical severity. Findings were confirmed in a validation cohort. Aim is that the score and haemocytometry results are simultaneously provided by analyser software, enabling wide applicability of the score as haemocytometry is commonly requested in COVID-19 patients

External Quality Assessment on Molecular Tumor Profiling with Circulating Tumor DNA-Based Methodologies Routinely Used in Clinical Pathology within the COIN Consortium

BACKGROUND: Identification of tumor-derived variants in circulating tumor DNA (ctDNA) has potential as a sensitive and reliable surrogate for tumor tissue-based routine diagnostic testing. However, variations in pre(analytical) procedures affect the efficiency of ctDNA recovery. Here, an external quality assessment (EQA) was performed to determine the performance of ctDNA mutation detection work flows that are used in current diagnostic settings across laboratories within the Dutch COIN consortium (ctDNA on the road to implementation in The Netherlands). METHODS: Aliquots of 3 high-volume diagnostic leukapheresis (DLA) plasma samples and 3 artificial reference plasma samples with predetermined mutations were distributed among 16 Dutch laboratories. Participating laboratories were requested to perform ctDNA analysis for BRAF exon 15, EGFR exon 18-21, and KRAS exon 2-3 using their regular circulating cell-free DNA (ccfDNA) analysis work flow. Laboratories were assessed based on adherence to the study protocol, overall detection rate, and overall genotyping performance. RESULTS: A broad range of preanalytical conditions (e.g., plasma volume, elution volume, and extraction methods) and analytical methodologies (e.g., droplet digital PCR [ddPCR], small-panel PCR assays, and next-generation sequencing [NGS]) were used. Six laboratories (38%) had a performance score of >0.90; all other laboratories scored between 0.26 and 0.80. Although 13 laboratories (81%) reached a 100% overall detection rate, the therapeutically relevant EGFR p.(S752_I759del) (69%), EGFR p.(N771_H773dup) (50%), and KRAS p.(G12C) (48%) mutations were frequently not genotyped accurately. CONCLUSIONS: Divergent (pre)analytical protocols could lead to discrepant clinical outcomes when using the same plasma samples. Standardization of (pre)analytical work flows can facilitate the implementation of reproducible liquid biopsy testing in the clinical routine

Validation of a new method for saliva cortisol testing to assess stress in first responders

Background Acute or chronic stress can lead to physical and mental disorders. Measuring cortisol can objectify the degree of stress. Cortisol is traditionally measured in serum, but recently the relevant fraction of free cortisol can be reliably measured in saliva, using the very sensitive liquid chromatography tandem mass spectrometry (LC-MS/MS) method. The use of saliva is non-invasive and allows easy serial testing around stressful events. The main objective of this study is to investigate whether serial saliva cortisol determinations using the LC-MS/MS method can be used to assess the stress response that first responders may experience during moments of acute professional deployment in their daily work. Methods Healthy first responders (police officers, firefighters, rapid response team, ambulance personnel, first aid and emergency medical personnel) were recruited to participate in a Euregional high-reliability simulation training ('Be Aware'-scenario training, 19 April 2018). At three time points, simultaneous venous blood samples and saliva samples were obtained. These time points were 1 hour before, immediately after and 10 hours after the simulation training. The correlation between changes in saliva cortisol measured by LC-MS/ MS and serum cortisol at all three time points was determined. Results were compared with spectators not directly participating in the simulation. Results 70 subjects participated in the simulation. There was a strong correlation between the changes in saliva and blood cortisol at the three time points. A significant increase in blood and saliva cortisol was shown 1 hour after the experienced stress moments. The levels had almost completely returned to baseline in all healthy volunteers 10 hours later. Cortisol in spectators was unaffected. Conclusion Serial saliva cortisol measurements using LC-MS/MS is a reliable and fast non-invasive functional stress assay, which can be easily collected in daily practice and used for investigation and monitoring of stress response in front line responders

Factor XIa and Thrombin Generation Are Elevated in Patients with Acute Coronary Syndrome and Predict Recurrent Cardiovascular Events

In acute coronary syndrome (ACS) cardiac cell damage is preceded by thrombosis. Therefore, plasma coagulation markers may have additional diagnostic relevance in ACS. By using novel coagulation assays this study aims to gain more insight into the relationship between the coagulation system and ACS.We measured plasma thrombin generation, factor XIa and D-dimer levels in plasma from ACS (n = 104) and non-ACS patients (n = 42). Follow-up measurements (n = 73) were performed at 1 and 6 months. Associations between coagulation markers and recurrent cardiovascular events were calculated by logistic regression analysis.Thrombin generation was significantly enhanced in ACS compared to non-ACS patients: peak height 148±53 vs. 122±42 nM. There was a significantly diminished ETP reduction (32 vs. 41%) and increased intrinsic coagulation activation (25 vs. 7%) in ACS compared to non-ACS patients. Furthermore, compared to non-ACS patients factor XIa and D-dimer levels were significantly elevated in ACS patients: 1.9±1.1 vs. 1.4±0.7 pM and 495(310-885) vs. 380(235-540) μg/L. Within the ACS spectrum, ST-elevated myocardial infarction patients had the highest prothrombotic profile. During the acute event, thrombin generation was significantly increased compared to 1 and 6 months afterwards: peak height 145±52 vs. 100±44 vs. 98±33 nM. Both peak height and factor XIa levels on admission predicted recurrent cardiovascular events (OR: 4.9 [95%CI 1.2-20.9] and 4.5 [1.1-18.9]).ACS patients had an enhanced prothrombotic profile, demonstrated by an increased thrombin generation potential, factor XIa and D-dimer levels. This study is the first to demonstrate the positive association between factor XIa, thrombin generation and recurrent cardiovascular events

A report on the potential of Rac1/pSTAT3 protein levels in T lymphocytes to assess the pharmacodynamic effect of thiopurine therapy in Inflammatory Bowel Disease patients

The thiopurine derivatives azathioprine (AZA), mercaptopurine (MP) and tioguanine (TG) remain standard treatment of inflammatory bowel disease (IBD). The immune suppressive effect of thiopurines is primarily based on blocking the Ras-related C3 botulinum toxin substrate 1 (Rac1) causing apoptosis of T lymphocytes by inhibition of the phosphorylated downstream transcription factor Signal Transducer and Activator of Transcription 3 (pSTAT3). A functional pharmacodynamic marker in T lymphocytes may be useful to predict therapeutic outcome of thiopurine therapy. The aim of this study was to explore whether protein levels of Rac1 and pSTAT3 in T lymphocytes may be applied as a specific pharmacodynamic marker for thiopurine therapy in IBD patients. Rac1 and pSTAT3 protein levels in T lymphocytes were explored in 57 IBD patients (median age 51 years, 56% female), subdivided into six groups based on IBD activity and its treatment: patients with active disease without IBD maintenance medication (1) or patients in remission on AZA/MP (2), TG (3), infliximab (IFX) (4), thiopurine and IFX combination-treatment (5) or without IBD medication (6). Reference values were obtained from healthy subjects. Rac1 and pSTAT3 protein levels in T lymphocytes from patients on thiopurine monotherapy (group 2 and 3) were compared to the other groups, and to healthy subjects. Absolute Rac1 and pSTAT3 protein levels showed no differences between the thiopurine monotherapy groups when compared to patients with active disease. However, the ratio of Rac1 and pSTAT3 protein levels was lower in thiopurine patients groups compared to patients with active disease. Rac1-corrected pSTAT3 protein levels may serve as a pharmacodynamic marker of thiopurine monotherapy and may be a potential tool to predict therapeutic effectiveness in IBD patients