Digestive Biomarkers of Laryngopharyngeal Reflux: A Preliminary Prospective Controlled Study.

OBJECTIVE: To investigate the digestive enzymes and biomarkers in the saliva of patients with laryngopharyngeal reflux (LPR) and asymptomatic individuals. STUDY DESIGN: Prospective controlled study. SETTING: Multicenter study. METHODS: Patients with LPR at the hypopharyngeal-esophageal impedance-pH monitoring (HEMII-pH) and asymptomatic individuals were consecutively recruited from January 2020 to April 2023 from 2 University Hospitals. The saliva of patients (off PPIs) and asymptomatic individuals was collected to measure pH, elastase, bile salts, cholesterol, gastric, and pancreatic lipases. Anxiety, symptoms, and findings were studied through perceived stress scale (PSS), reflux symptom score (RSS), and reflux sign assessment (RSA). RESULTS: Sixty-seven LPR patients and 57 asymptomatic individuals completed the evaluations. LPR patients reported higher PSS, RSS, and RSA than asymptomatic individuals. The mean saliva pH was more alkaline in LPR patients (7.23: 95% confidence interval [CI]: 7.08, 7.38) compared to controls (6.13; 95% CI: 5.95, 6.31; P = .001). The mean concentration of elastase was higher in patients (51.65 µg/mL; 95% CI: 44.47, 58.83 µg/mL) versus asymptomatic individuals (25.18 µg/mL; 95% CI: 21.64, 28.72 µg/mL; P = .001). The saliva cholesterol reported higher concentration in healthy individuals (3.43 mg/dL; 95% CI: 3.21, 3.65 mg/dL) compared to patients (1.16 mg/dL; 95% CI: 1.05, 1.27 mg/dL; P = .001). The saliva pH, and elastase concentration were significantly associated with the baseline RSS, while saliva cholesterol was negatively associated with the severity of RSS and RSA. CONCLUSION: Cholesterol, bile salts, and elastase are biomarkers of LPR and should be considered to develop future non-invasive saliva device for the detection of LPR.

Pulmonary Functional Outcomes at 3 Months in Critical COVID-19 Survivors Hospitalized during the First, Second, and Third Pandemic Waves.

INTRODUCTION: Despite improved management of patients with COVID-19, we still ignore whether pharmacologic treatments and improved respiratory support have modified outcomes for intensive care unit (ICU) surviving patients of the three first consecutive waves (w) of the pandemic. The aim of this study was to evaluate whether developments in the management of ICU COVID-19 patients have positively impacted respiratory functional outcomes, quality of life (QoL), and chest CT scan patterns in ICU COVID-19 surviving patients at 3 months, according to pandemic waves. METHODS: We prospectively included all patients admitted to the ICU of two university hospitals with acute respiratory distress syndrome (ARDS) related to COVID-19. Data related to hospitalization (disease severity, complications), demographics, and medical history were collected. Patients were assessed 3 months post-ICU discharge using a 6 min walking distance test (6MWT), a pulmonary function test (PFT), a respiratory muscle strength (RMS) test, a chest CT scan, and a Short Form 36 (SF-36) questionnaire. RESULTS: We included 84 ARDS COVID-19 surviving patients. Disease severity, complications, demographics, and comorbidities were similar between groups, but there were more women in wave 3 (w3). Length of stay at the hospital was shorter during w3 vs. during wave 1 (w1) (23.4 ± 14.2 days vs. 34.7 ± 20.8 days, p = 0.0304). Fewer patients required mechanical ventilation (MV) during the second wave (w2) vs. during w1 (33.3% vs. 63.9%, p = 0.0038). Assessment at 3 months after ICU discharge revealed that PFTs and 6MWTs scores were worse for w3 > w2 > w1. QoL (SF-36) deteriorated (vitality and mental health) more for patients in w1 vs. in w3 (64.7 ± 16.3 vs. 49.2 ± 23.2, p = 0.0169). Mechanical ventilation was associated with reduced forced expiratory volume (FEV1), total lung capacity (TLC), diffusing capacity for carbon monoxide (DLCO), and respiratory muscle strength (RMS) (w1,2,3, p < 0.0500) on linear/logistic regression analysis. The use of glucocorticoids, as well as tocilizumab, was associated with improvements in the number of affected segments in chest CT, FEV1, TLC, and DLCO (p < 0.01). CONCLUSIONS: With better understanding and management of COVID-19, there was an improvement in PFT, 6MWT, and RMS in ICU survivors 3 months after ICU discharge, regardless of the pandemic wave during which they were hospitalized. However, immunomodulation and improved best practices for the management of COVID-19 do not appear to be sufficient to prevent significant morbidity in critically ill patients.

Determination of phage susceptibility as a clinical diagnostic tool: A routine perspective.

As the global burden of disease caused by multidrug resistant bacteria is a major source of concern, credible clinical alternatives to antibiotic therapy, such as personalized phage therapy, are actively explored. Although phage therapy has been used for more than a century, the issue of an easy to implement diagnostic tool for determining phage susceptibility that meets current routine clinical needs is still open. In this Review, we summarize the existing methods used for determining phage activity on bacteria, including the three reference methods: the spot test, the double agar overlay plaque assay, and the Appelmans method. The first two methods rely on the principle of challenging the overnight growth of a lawn of bacteria in an agar matrix to a known relative phage to bacteria concentration and represent good screening tools to determine if the tested phage can be used for a "passive" and or "active" treatment. Beside these methods, several techniques, based on "real-time" growth kinetics assays (GKA) have been developed or are under development. They all monitor the growth of clinical isolates in the presence of phages, but use various detection methods, from classical optical density to more sophisticated techniques such as computer-assisted imagery, flow-cytometry, quantitative real-time polymerase chain reaction (qPCR) or metabolic indicators. Practical considerations as well as information provided about phage activity are reviewed for each technique. Finally, we also discuss the analytical and interpretative requirements for the implementation of a phage susceptibility testing tool in routine clinical microbiology.

Chemerin plasma levels are increased in COVID-19 patients and are an independent risk factor of mortality.

Background: Chemerin is an extracellular protein with chemotactic activities and its expression is increased in various diseases such as metabolic syndrome and inflammatory conditions. Its role in lung pathology has not yet been extensively studied but both known pro- and anti-inflammatory properties have been observed. The aim of our study was to evaluate the involvement of the chemerin/ChemR23 system in the physiopathology of COVID-19 with a particular focus on its prognostic value. Methods: Blood samples from confirmed COVID-19 patients were collected at day 1, 5 and 14 from admission to Erasme Hospital (Brussels - Belgium). Chemerin concentrations and inflammatory biomarkers were analyzed in the plasma. Blood cells subtypes and their expression of ChemR23 were determined by flow cytometry. The expression of chemerin and ChemR23 was evaluated on lung tissue from autopsied COVID-19 patients by immunohistochemistry (IHC). Results: 21 healthy controls (HC) and 88 COVID-19 patients, including 40 in intensive care unit (ICU) were included. Plasma chemerin concentration were significantly higher in ICU patients than in HC at all time-points analyzed (p<0.0001). Moreover, they were higher in deceased patients compared to survivors (p<0.05). Logistic univariate regression and multivariate analysis demonstrated that chemerin level at day 14 of admission was an independent risk factor for death. Accordingly, chemerin levels correlated with inflammatory biomarkers such as C-reactive protein and tumor necrosis factor α. Finally, IHC analysis revealed a strong expression of ChemR23 on smooth muscle cells and chemerin on myofibroblasts in advanced acute respiratory distress syndrome (ARDS). Discussion: Increased plasma chemerin levels are a marker of severity and may predict death of COVID-19 patients. However, multicentric studies are needed, before chemerin can be considered as a biomarker of severity and death used in daily clinical practice. Further studies are also necessary to identify the precise mechanisms of the chemerin/ChemR23 system in ARDS secondary to viral pneumonia.

Organisation and quality monitoring for point-of-care testing (POCT) in Belgium: proposal for an expansion of the legal framework for POCT into primary health care.

BACKGROUND: There is a trend towards decentralisation of laboratory tests by means of Point-of-Care testing (POCT). Within hospitals, Belgian law requires a POCT policy, coordinated by the clinical laboratory. There is however no legal framework for POCT performed outside the hospital: no reimbursement, no compulsory quality monitoring and no limits nor control on the prices charged to the patient. Uncontrolled use of POCT can have negative consequences for individual and public health. PROPOSAL: We propose that POCT outside hospitals would only be reimbursed for tests carried out within a legal framework, requiring evidence-based testing and collaboration with a clinical laboratory, because clinical laboratories have procedures for test validation and quality monitoring, are equipped for electronic data transfer, are familiar with logistical processes, can provide support when technical issues arise and can organise and certify training. Under these conditions the government investment will be offset by health benefits, e.g. fall in antibiotic consumption with POCT for CRP in primary care, quick response to SARS-CoV2-positive cases in COVID-19 triage centres. PRIORITIES: 1° extension of the Belgian decree on certification of clinical laboratories to decentralised tests in primary care; 2° introduction of a separate reimbursement category for POCT; 3° introduction of reimbursement for a limited number of specified POCT; 4° setup of a Multidisciplinary POCT Advisory Council, the purpose of which is to draw up a model for reimbursement of POCT, to select tests eligible for reimbursement and to make proposals to the National Institute for Health and Disability Insurance (RIZIV/INAMI).

Development and validation of a simple correction method for the measurement of neuron-specific enolase in hemolyzed serum samples.

The Neuron-specific enolase (NSE), a biomarker of neuroendocrine tumors or ischemic brain damage, has limited clinical applicability since its measurement is overestimated by hemolysis. In this study, an NSE correction method was developed for hemolyzed samples. The NSE concentration and the hemolysis index (HI) of serum were measured before and after spiking a hemolysate prepared with red blood cells from the serum-separating tube and extrapolating the NSE value corresponding to a HI of zero. To validate the approach (n = 46), NSE concentrations and HI were measured before (NSE0 and HI0) and after spiking the samples with 50 µL (HIA, NSEA) and 100 µL (HIB, NSEB) of hemolysate. A linear regression analysis was performed between (HIA, NSEA) and (HIB, NSEB). The y-intercept was taken as the corrected NSE concentration (NSEintercept) and compared with NSE0. On the same samples, the equation of Tolan et al. was applied and the corrected values of NSE (NSEcorr) were compared to NSE0. The average bias (±SD) between the NSE0 and the NSEintercept was equal to -3.2% (± 14.3) versus 34.6% (± 19.8) against the NSEcorr. Applying the allowable total error proposed by the European Federation of Laboratory Medicine, 72% of the NSE results were adequately corrected while the reference method corrected only 8.7% of the results. The individualized hemolysis correction method developed is simple, fast, requires one serum-separating tube, provides increased accuracy compared to the method described by Tolan et al. and should improve the quality of patient care.

SARS-CoV-2 Diagnostic Tests: Algorithm and Field Evaluation From the Near Patient Testing to the Automated Diagnostic Platform.

Introduction: Since the first wave of COVID-19 in Europe, new diagnostic tools using antigen detection and rapid molecular techniques have been developed. Our objective was to elaborate a diagnostic algorithm combining antigen rapid diagnostic tests, automated antigen dosing and rapid molecular tests and to assess its performance under routine conditions. Methods: An analytical performance evaluation of four antigen rapid tests, one automated antigen dosing and one molecular point-of-care test was performed on samples sent to our laboratory for a SARS-CoV-2 reverse transcription PCR. We then established a diagnostic algorithm by approaching median viral loads in target populations and evaluated the limit of detection of each test using the PCR cycle threshold values. A field performance evaluation including a clinical validation and a user-friendliness assessment was then conducted on the antigen rapid tests in point-of-care settings (general practitioners and emergency rooms) for outpatients who were symptomatic for <7 days. Automated antigen dosing was trialed for the screening of asymptomatic inpatients. Results: Our diagnostic algorithm proposed to test recently symptomatic patients using rapid antigen tests, asymptomatic patients using automated tests, and patients requiring immediate admission using molecular point-of-care tests. Accordingly, the conventional reverse transcription PCR was kept as a second line tool. In this setting, antigen rapid tests yielded an overall sensitivity of 83.3% (not significantly different between the four assays) while the use of automated antigen dosing would have spared 93.5% of asymptomatic inpatient screening PCRs. Conclusion: Using tests not considered the "gold standard" for COVID-19 diagnosis on well-defined target populations allowed for the optimization of their intrinsic performances, widening the scale of our testing arsenal while sparing molecular resources for more seriously ill patients.

Post-discharge critical COVID-19 lung function related to severity of radiologic lung involvement at admission.

Lung function impairment persists in 55% of critical COVID-19 patients three months after ICU discharge. Patient lung function, exercise capacity, radiologic, and quality of life data suggest impairment is related to radiologic lung involvement at admission.

Laryngopharyngeal reflux: The microbiota theory.

Laryngopharyngeal reflux (LPR) is a prevalent disease associated with non-specific symptoms and findings. Many gray areas persist in the pathogenesis of LPR, the diagnosis and the treatment. Symptoms are poorly correlated with fiberoptic signs or hypopharyngeal-esophageal multichannel intraluminal impedance-pH monitoring findings. The therapeutic response remains uncertain with some resistant patients to medical or surgical treatment. The development of LPR-symptoms and findings may be related to the refluxate of a myriad of gastroduodenal enzymes, which may modify the laryngopharyngeal and oral microbiome leading to mucosa maintenance and recovery impairments. The diet of patient is important because it may impact the microbiome composition and some foods are known to increase the number of hypopharyngeal reflux events. The number of hypopharyngeal reflux events may be increased by autonomic nerve dysfunction that may have an important role in the persistence of LPR-symptoms.

Clinical Assessment of Breast Volume: Can 3D Imaging Be the Gold Standard?

Three-dimensional (3D) camera systems are increasingly used for computerized volume calculations. In this study we investigate whether the Vectra XT 3D imaging system is a reliable tool for determination of breast volume in clinical practice. It is compared with the current gold standard in literature, magnetic resonance imaging (MRI), and current clinical practice (plastic surgeon's clinical estimation). METHODS: Breast volumes of 29 patients (53 breasts) were evaluated. 3D images were acquired by Vectra XT 3D imaging system. Pre-existing breast MRI images were collected. Both imaging techniques were used for volume analyses, calculated by two independent investigators. Breast volume estimations were done by plastic surgeons during outpatient consultations. All volume measurements were compared using paired samples t-test, intra-class correlation coefficient, Pearson's correlation, and Bland-Altman analysis. RESULTS: Two 3D breast volume measurements showed an excellent reliability (intra-class correlation coefficient: 0.991), which was comparable to the reliability of MRI measurements (intra-class correlation coefficient: 0.990). Mean (SD) breast volume measured with 3D breast volume was 454 cm3 (157) and with MRI was 687 cm3 (312). These volumes were significantly different, but a linear association could be found: y(MRI) = 1.58 × (3D) - 40. Three-dimensional breast volume was not significantly different from volume estimation made by plastic surgeons (472 cm3 (69), P = 0.323). CONCLUSIONS: The 3D imaging system measures lower volumes for breasts than MRI. However, 3D measurements show a linear association with MRI and have excellent reliability, making them an objective and reproducible measuring method suitable for clinical practice.

Development and Potential Usefulness of the COVID-19 Ag Respi-Strip Diagnostic Assay in a Pandemic Context.

Introduction: COVID-19 Ag Respi-Strip, an immunochromatographic (ICT) assay for the rapid detection of SARS-CoV-2 antigen on nasopharyngeal specimen, has been developed to identify positive COVID-19 patients allowing prompt clinical and quarantine decisions. In this original research article, we describe the conception, the analytical and clinical performances as well as the risk management of implementing the COVID-19 Ag Respi-Strip in a diagnostic decision algorithm. Materials and Methods: Development of the COVID-19 Ag Respi-Strip resulted in a ready-to-use ICT assay based on a membrane technology with colloidal gold nanoparticles using monoclonal antibodies directed against the SARS-CoV and SARS-CoV-2 highly conserved nucleoprotein antigen. Four hundred observations were recorded for the analytical performance study and thirty tests were analyzed for the cross-reactivity study. The clinical performance study was performed in a retrospective multi-centric evaluation on aliquots of 328 nasopharyngeal samples. COVID-19 Ag Respi-Strip results were compared with qRT-PCR as golden standard for COVID-19 diagnostics. Results: In the analytical performance study, the reproducibility showed a between-observer disagreement of 1.7%, a robustness of 98%, an overall satisfying user friendliness and no cross-reactivity with other virus-infected nasopharyngeal samples. In the clinical performance study performed in three different clinical laboratories during the ascendant phase of the epidemiological curve, we found an overall sensitivity and specificity of 57.6 and 99.5%, respectively with an accuracy of 82.6%. The cut-off of the ICT was found at CT <22. User-friendliness analysis and risk management assessment through Ishikawa diagram demonstrate that COVID-19 Ag Respi-Strip may be implemented in clinical laboratories according to biosafety recommendations. Conclusion: The COVID-19 Ag Respi-Strip represents a promising rapid SARS-CoV-2 antigen assay for the first-line diagnosis of COVID-19 in 15 min at the peak of the pandemic. Its role in the proposed diagnostic algorithm is complementary to the currently-used molecular techniques.

The pathway through LC-MS method development: in-house or ready-to-use kit-based methods?

Historically, the determination of low concentration analytes was initially made possible by the development of rapid and easy-to-perform immunoassays (IAs). Unfortunately, typical problems inherent to IA technologies rapidly appeared (e.g. elevated cost, cross-reactivity, lot-to-lot variability, etc.). In turn, liquid chromatography tandem mass spectrometry (LC-MS/MS) methods are sensitive and specific enough for such analyses. Therefore, they would seem to be the most promising candidates to replace IAs. There are two main choices when implementing a new LC-MS/MS method in a clinical laboratory: (1) Developing an in-house method or (2) purchasing ready-to-use kits. In this paper, we discuss some of the respective advantages, disadvantages and mandatory requirements of each choice. Additionally, we also share our experiences when developing an in-house method for cortisol determination and the implementation of an "ready-to-use" (RTU) kit for steroids analysis.