Molecular allergology: a clinical laboratory tool for precision diagnosis, stratification and follow-up of allergic patients.

Identification of the molecular culprits of allergic reactions leveraged molecular allergology applications in clinical laboratory medicine. Molecular allergology shifted the focus from complex, heterogeneous allergenic extracts, e.g. pollen, food, or insect venom, towards genetically and immunologically defined proteins available for in vitro diagnosis. Molecular allergology is a precision medicine approach for the diagnosis, stratification, therapeutic management, follow-up and prognostic evaluation of patients within a large range of allergic diseases. Exclusively available for in vitro diagnosis, molecular allergology is nonredundant with any of the current clinical tools for allergy investigation. As an example of a major application, discrimination of genuine sensitization from allergen cross-reactivity at the molecular level allows the proper targeting of the culprit allergen and thus dramatically improves patient management. This review aims at introducing clinical laboratory specialists to molecular allergology, from the biochemical and genetic bases, through immunological concepts, to daily use in the diagnosis and management of allergic diseases.

Practices of French General Practitioners Regarding Vaccination of Boys Against Human Papillomavirus (HPV), One Year After the Application of Its Official Recommendation.

In 2019, French health authorities extended the recommendation for human papillomavirus (HPV) vaccination to include boys aged 11 to 19 years. We describe HPV vaccination practices among French general practitioners (GPs) since this recommendation wasapplied. We also identified factors associated with the propensity to propose HPV vaccination to boys. Cross-sectional study, between May and August 2022, among French GPs using a questionnaire asking about the GPs, their practices, and opinions regarding HPV vaccination, including whether they systematically proposed HPV vaccination to eligible boys or not. We investigated factors associated with systematic proposal of HPV vaccination, using univariate and multivariate logistic regression. In total, 360 GPs participated (76.6% females; mean age 34.7 ± 7.8 years; 22.9% had additional training in gynecology or pediatrics); 5.5% reported that they systematically offered HPV vaccination to boys prior to the recommendation, whereas 61.2% do so systematically since the recommendation. Factors associated with systematic proposal to boys (post recommendation) were female GP sex (78.6% versus 66.2%; OR = 2.0 [95% confidence interval (CI) 1.2-3.3]; p = 0.007) and systematic proposal prior to the recommendation (8.5% versus 0.7%; OR = 13.3 [1.7-101.7]; p = 0.01). Protection against HPV-induced cancer was cited as an argument to vaccinate girls (98.3% versus 89.2%; p < 0.0001); while reducing the risk of transmission was more commonly an argument to vaccinate boys (78.1% versus 51.8%; p < 0.0001). This study underlines the positive impact of the official recommendation for HPV vaccination of boys on the attitude of GPs, with an increase in the systematic proposal of HPV vaccination to boys.

Early plasma interferon-ß levels as a predictive marker of COVID-19 severe clinical events in adult patients.

We assessed relationships between early peripheral blood type I interferons (IFN) levels, clinical new early warning scores (NEWS), and clinical outcomes in hospitalized coronavirus disease-19 (COVID-19) adult patients. Early IFN-ß levels were lower among patients who further required intensive care unit (ICU) admission than those measured in patients who did not require an ICU admission during severe acute respiratory syndrome coronavirus type 2 infection. IFN-ß levels were inversely correlated with NEWS only in the subgroup of patients who further required ICU admission. To assess whether peripheral blood IFN-ß levels could be a potential relevant biomarker to predict further need for ICU admission, we performed receiver operating characteristic (ROC) curve analyses that showed for all study patients an area under ROC curve of 0.77 growing to 0.86 (p = 0.003) when the analysis was restricted to a subset of patients with NEWS ≥5 at the time of hospital admission. Overall, our findings indicated that early peripheral blood IFN-ß levels might be a relevant predictive marker of further need for an ICU admission in hospitalized COVID-19 adult patients, specifically when clinical score (NEWS) was graded as upper than 5 at the time of hospital admission.

Impact of a dTcaP booster vaccine awareness campaign initiated by the French national health insurance for adults aged 25 years in 2021.

BACKGROUND: Vaccination schedules differ from country to country. In France, the diphtheria, tetanus, pertussis, poliomyelitis (dTcaP) booster vaccine coverage for adults aged 25 has been lower than those recommended. We evaluated the impact of an awareness campaign undertaken by the French national health insurance system in 2021. METHODS: A randomized, controlled study with adults residing in the Ardennes region was conducted to evaluate the effect on vaccine coverage of the booster vaccine reminder campaign carried out via letter and/or email and/or SMS. The randomization unit was the municipal administrative area (canton). Ten cantons were grouped into the intervention group (INT) and nine were the control group (CON). Outcomes were the booster vaccine delivery and the consultation of a general practitioner (GP) within 12 months (since the French national health insurance running the campaign suggested patients to consult their GP). RESULTS: A total of 1,975 adults were included (INT: 67.3% vs. CON: 32.7%). Of them, 331 received a booster vaccine (INT: 17.4% vs. CON: 15.5%; p = 0.29), and 1,442 consulted a GP (INT: 73.7% vs. CON: 76.8%; p = 0.14). Those who consulted a GP had more frequent vaccine delivery (INT: 19.1% vs. CON: 10.5%; p < 0.0001). CONCLUSIONS: This study found that the awareness campaign run by the French national health insurance did not improve the uptake of the dTcaP booster and that there was a low rate of vaccinated adults aged 25 years. A GP consultation was associated with dTcaP booster vaccine delivery which may show that there is a need of involving GPs in vaccination follow-ups. Patients recognize GPs as providers of credible information and they may play a key role in individualized preventive healthcare actions. Systematic consultations with GPs for follow-up could be proposed to insured adults aged 25 years in the future.

The COVIDTW study: Clinical predictors of COVID-19 mortality and a novel AI prognostic model using chest X-ray.

BACKGROUND: There is a lack of published research on the impact of the first wave of the COVID-19 pandemic in Taiwan. We investigated the mortality risk factors among critically ill patients with COVID-19 in Taiwan during the initial wave. Furthermore, we aim to develop a novel AI mortality prediction model using chest X-ray (CXR) alone. METHOD: We retrospectively reviewed the medical records of patients with COVID-19 at Taipei Tzu Chi Hospital from May 15 to July 15 2021. We enrolled adult patients who received invasive mechanical ventilation. The CXR images of each enrolled patient were divided into 4 categories (1st, pre-ETT, ETT, and WORST). To establish a prediction model, we used the MobilenetV3-Small model with "Imagenet" pretrained weights, followed by high Dropout regularization layers. We trained the model with these data with Five-Fold Cross-Validation to evaluate model performance. RESULT: A total of 64 patients were enrolled. The overall mortality rate was 45%. The median time from symptom onset to intubation was 8 days. Vasopressor use and a higher BRIXIA score on the WORST CXR were associated with an increased risk of mortality. The areas under the curve of the 1st, pre-ETT, ETT, and WORST CXRs by the AI model were 0.87, 0.92, 0.96, and 0.93 respectively. CONCLUSION: The mortality rate of COVID-19 patients who receive invasive mechanical ventilation was high. Septic shock and high BRIXIA score were clinical predictors of mortality. The novel AI mortality prediction model using CXR alone exhibited a high performance.

Electrocardiogram Heartbeat Classification for Arrhythmias and Myocardial Infarction.

An electrocardiogram (ECG) is a basic and quick test for evaluating cardiac disorders and is crucial for remote patient monitoring equipment. An accurate ECG signal classification is critical for real-time measurement, analysis, archiving, and transmission of clinical data. Numerous studies have focused on accurate heartbeat classification, and deep neural networks have been suggested for better accuracy and simplicity. We investigated a new model for ECG heartbeat classification and found that it surpasses state-of-the-art models, achieving remarkable accuracy scores of 98.5% on the Physionet MIT-BIH dataset and 98.28% on the PTB database. Furthermore, our model achieves an impressive F1-score of approximately 86.71%, outperforming other models, such as MINA, CRNN, and EXpertRF on the PhysioNet Challenge 2017 dataset.

Expanding the Molecular Alphabet of DNA-Based Data Storage Systems with Neural Network Nanopore Readout Processing.

DNA is a promising next-generation data storage medium, but challenges remain with synthesis costs and recording latency. Here, we describe a prototype of a DNA data storage system that uses an extended molecular alphabet combining natural and chemically modified nucleotides. Our results show that MspA nanopores can discriminate different combinations and ordered sequences of natural and chemically modified nucleotides in custom-designed oligomers. We further demonstrate single-molecule sequencing of the extended alphabet using a neural network architecture that classifies raw current signals generated by Oxford Nanopore sequencers with an average accuracy exceeding 60% (39× larger than random guessing). Molecular dynamics simulations show that the majority of modified nucleotides lead to only minor perturbations of the DNA double helix. Overall, the extended molecular alphabet may potentially offer a nearly 2-fold increase in storage density and potentially the same order of reduction in the recording latency, thereby enabling new implementations of molecular recorders.

An Engineered OmpG Nanopore with Displayed Peptide Motifs for Single-Molecule Multiplex Protein Detection.

Molecular detection via nanopore, achieved by monitoring changes in ionic current arising from analyte interaction with the sensor pore, is a promising technology for multiplex sensing development. Outer Membrane Protein G (OmpG), a monomeric porin possessing seven functionalizable loops, has been reported as an effective sensing platform for selective protein detection. Using flow cytometry to screen unfavorable constructs, we identified two OmpG nanopores with unique peptide motifs displayed in either loop 3 or 6, which also exhibited distinct analyte signals in single-channel current recordings. We exploited these motif-displaying loops concurrently to facilitate single-molecule multiplex protein detection in a mixture. We additionally report a strategy to increase sensor sensitivity via avidity motif display. These sensing schemes may be expanded to more sophisticated designs utilizing additional loops to increase multiplicity and sensitivity.

A Nanopore Approach for Analysis of Caspase-7 Activity in Cell Lysates.

Caspases are an important protease family that coordinate inflammation and programmed cell death. Two closely related caspases, caspase-3 and caspase-7, exhibit largely overlapping substrate specificities. Assessing their proteolytic activities individually has therefore proven extremely challenging. Here, we constructed an outer membrane protein G (OmpG) nanopore with a caspase substrate sequence DEVDG grafted into one of the OmpG loops. Cleavage of the substrate sequence in the nanopore by caspase-7 generated a characteristic signal in the current recording of the OmpG nanopore that allowed the determination of the activity of caspase-7 in Escherichia coli cell lysates. Our approach may provide a framework for the activity-based profiling of proteases that share highly similar substrate specificity spectrums.

Effect of Diet in Chronic Spontaneous Urticaria: A Systematic Review.

Strategies for diets in chronic spontaneous urticaria (CSU) are controversial. This systematic review assessed the interest in diet for managing CSU. We searched for original reports in MEDLINE, EMBASE, CENTRAL and LILACS. Among the 278 reports screened, 20 were included, involving 1,734 patients. Reports described 3 types of systematic diet: pseudoallergen-free diet (n = 1,555 patients), low-histamine diet (n = 223) and diet without fish products (n = 47), which induced complete remission in 4.8%, 11.7% and 10.6% of patients, respectively, and partial remission in 37.0%, 43.9% and 4.3%. Eight reports described personalized exclusion diets (66 patients) adapted to symptoms/allergological test results and led to complete remission in 74.6% of patients, although the diagnosis of CSU was doubtful. No comparative randomized studies of diets were available. The only randomized studies were based on oral provocation tests with the suspected responsible diet. Population and outcomes were heterogeneous. In conclusion, there is evidence for the benefit of diets in CSU only in individual patients with clinical symptoms. However, the level of evidence is low for the benefit of systematic diets in CSU because systematic double-blind controlled trials of diet are lacking.

Chronic spontaneous urticaria in children - a systematic review on interventions and comorbidities.

BACKGROUND: Chronic spontaneous urticaria (CSU) is not frequent in children. Management guidelines have been developed for adults and randomized controlled trials (RCTs) included teenagers aged 12-18, but data for children under age 12 are limited. We performed a systematic review to assess comorbidities in children <12 years old with CSU and the efficacy and safety of treatments. METHODS: We searched for original articles of epidemiologic and treatment data in children <12 years old with CSU that were published from 2005 to July 2016 in MEDLINE, EMBASE, CENTRAL, and LILACS. Article selection and data extraction were performed in duplicate. RESULTS: Our systematic review included 9 reports on epidemiologic data (633 children). Five comorbidities and laboratory anomalies associated with CSU found were atopy (28.1%), positive autologous serum skin test (36.8%), thyroid biologic anomalies (6.4%) and detectable antinuclear antigen (10.4%), seroprevalence for Helicobacter pylori (21.1%), low vitamin D level (69.1%), and psychiatric disorders (70.4%). Only one study allowed for comparison with a control group. Our review included 10 studies (322 children), describing 5 different drug families, mostly H1-antihistamines (n = 297). One randomized controlled study compared single-dose rupatadine with single-dose desloratadine and placebo. Cyclosporine was effective and had no adverse effects in 18 children. Omalizumab, montelukast, and cefuroxime were reported in very small series (5, 1, and 1 patients). CONCLUSIONS: H1-antihistamines are effective for CSU in children <12 years old, with reassuring safety data at licensed doses. Cyclosporine seems effective, but the level of evidence is low.

Interrogating Detergent Desolvation of Nanopore-Forming Proteins by Fluorescence Polarization Spectroscopy.

Understanding how membrane proteins interact with detergents is of fundamental and practical significance in structural and chemical biology as well as in nanobiotechnology. Current methods for inspecting protein-detergent complex (PDC) interfaces require high concentrations of protein and are of low throughput. Here, we describe a scalable, spectroscopic approach that uses nanomolar protein concentrations in native solutions. This approach, which is based on steady-state fluorescence polarization (FP) spectroscopy, kinetically resolves the dissociation of detergents from membrane proteins and protein unfolding. For satisfactorily solubilizing detergents, at concentrations much greater than the critical micelle concentration (CMC), the fluorescence anisotropy was independent of detergent concentration. In contrast, at detergent concentrations comparable with or below the CMC, the anisotropy readout underwent a time-dependent decrease, showing a specific and sensitive protein unfolding signature. Functionally reconstituted membrane proteins into a bilayer membrane confirmed predictions made by these FP-based determinations with respect to varying refolding conditions. From a practical point of view, this 96-well analytical approach will facilitate a massively parallel assessment of the PDC interfacial interactions under a fairly broad range of micellar and environmental conditions. We expect that these studies will potentially accelerate research in membrane proteins pertaining to their extraction, solubilization, stabilization, and crystallization, as well as reconstitution into bilayer membranes.

Biomarkers related to bullous pemphigoid activity and outcome.

Bullous pemphigoid (BP) is the most common autoimmune subepidermal blistering disease of the skin. Investigation of the BP-associated pathophysiological processes during the last decades showed that the generation of autoantibodies directed against the hemidesmosome proteins BP180 and BP230, a hallmark of the BP-associated autoimmune response, leads to the recruitment of inflammatory immune cells at the dermal-epidermal junction, and subsequently to the release of a large amount of inflammatory molecules involved in blister formation. Analysis in transversal and longitudinal studies of autoantibodies and inflammatory molecules production both at the time of diagnosis and under treatment was mainly performed within the serum but also in the blister fluid. Some autoimmune or inflammatory molecules expression was related to the presence of clinical signs, while others were mere bystanders. In this review, we focused on the autoimmune and inflammatory molecules that have been identified as potential biomarkers of BP development and outcome.

Positive Direct Immunofluorescence Is of Better Value than ELISA-BP180 and ELISA-BP230 Values for the Prediction of Relapse after Treatment Cessation in Bullous Pemphigoid: A Retrospective Study of 97 Patients.

BACKGROUND: ELISA-BP180 values and direct immunofluorescence (DIF) are prognostic factors for relapse after treatment cessation in bullous pemphigoid (BP). OBJECTIVE: To determine the relevance of ELISA-BP230 antibodies for predicting relapse 6 months after treatment cessation. METHODS: We retrospectively selected patients with BP and available data from ELISA-BP180 and -BP230 and DIF performed at treatment cessation. The rate of relapse was calculated at 6 months. We compared ELISA-BP180 and -BP230 values and DIF in patients with relapse and remission. RESULTS: We included 97 patients. At 6 months, 25.6% of patients showed relapse. The proportion of patients with an ELISA-BP230 value ≥27 UA/ml was higher, but not significantly, for those with relapse than for those with remission (p = 0.11). The frequency of positive DIF findings was significantly higher for patients with relapse (p = 0.005). CONCLUSION: DIF is of better value than ELISA-BP180 and -230 tests to predict relapse after treatment cessation in BP.

Reinforcement during the COVID-19 pandemic: Perception of nursing students and impact on intention to drop-out of nursing education.

Background: Although the COVID-19 pandemic's impact on students has already been studied, its impact on nursing students' perception of their training and their conception of their future profession is unknown. Aims: To describe nursing students' perception of their involvement in reinforcement during the COVID-19 pandemic and the impact of working as reinforcement staff during the COVID-19 pandemic on nursing students. Design: Cross-sectional, comparative case/non-case study. Setting: nurse training institutions in France. Participants: "Cases" defined as nursing students who worked as reinforcement staff during the COVID-19 pandemic; "non-cases" defined as people who were in final year of nursing studies in 2018-2019 or 2019-2020 and so did not work as reinforcement staff during their nursing studies. Methods: questionnaire about representations of the nursing profession, role of the nurse in society, previous thinking of dropping out of nursing education. Results: 534 subjects included (310 cases; 214 non-cases). Cases reported feeling useful (38.6%) or very useful (25.7%) as reinforcement workers, while 91.5% concurred that nurses had an important role in the management of COVID-19 patients. Cases more frequently reported that the nursing profession is one where you save lives (61.5% vs 52.5%, p = 0.05). The desire to work as a nurse for a whole life had been more frequently expressed by cases (45.3% vs 34.8%, p = 0.05). Nursing education drop-out has been considered by 63.4% of subjects, without difference between "cases" and "non-cases" (p = 0.63). Subjects who considered dropping out of nursing education were younger (p = 0.01) and less often prone to think that the nursing profession was a profession personally rewarding (p = 0.01) and a life-saving profession (p = 0.03). Conclusion: The majority of nursing students reported feeling useful during the pandemic, and underlined the importance of the nurse's role in management of COVID-19 patients. Participation in reinforcement staff during the pandemic had no influence on dropping out of nursing education.

Molecular background of novel silent RHCE alleles.

BACKGROUND: The absence of expression of C/c and E/e antigens has been associated with rare variant RHCE alleles, referred to as silent RHCE alleles, classically identified among individuals with a rare D- - or Rhnull phenotype. This work reports on different molecular mechanisms identified in three novel silent RHCE alleles. STUDY DESIGN AND METHODS: Samples from D- - or Rhnull individuals and their family members, from families for whom Rh phenotype and/or serologic data were unexplained by inheritance of conventional RH alleles, were analyzed. Genomic DNA and transcripts were tested by sequencing analysis. RESULTS: The first silent allele was a RHCE*cE allele carrying an intronic IVS3+5G>A mutation. The second was a RHCE*ce allele carrying an intronic IVS7-2A>G mutation, whereas the third was a silent RHCE*ce allele carrying a 5-bp deletion (Nucleotides 679-683) in Exon 5. CONCLUSION: In addition to hybrid alleles and nucleotide deletion, intronic mutations may be associated with the nonexpression of RhCE antigens. Regarding the RH system, silent alleles may not be investigated among D- - or Rhnull individuals only. Rh phenotype and/or serologic data unexplained by inheritance of conventional RH alleles should lead to molecular investigations.

Identification of novel silent KEL alleles causing KEL:-5 (Ko) phenotype or discordance between KEL:1,-2 phenotype/KEL*01/02 genotype.

BACKGROUND: The Kell system, encoded by the KEL gene, is one of the most clinically important blood group systems. Molecular defects may lead to the absence of Kell antigen expression. The very rare KEL:5 results from silent KEL genes, also called KELnull alleles. In a few cases, the rare KEL:1,-2 phenotype may be associated with silent KEL*02 alleles. STUDY DESIGN AND METHODS: The aim of this study was to perform DNA investigations to identify silent KEL alleles among 10 KEL:-5 patients and 121 individuals presenting the rare KEL:1,-2 phenotype. Serologic investigations were performed on patients' red blood cells and serum. The KEL gene analysis was done by using a BeadChip assay (HEA Version, 1.2, Immucor), real-time polymerase chain reaction, and/or sequencing of all 19 exons of the KEL gene. RESULTS: In KEL:-5 patients, two novel KELnull alleles were described: 821G>A being the second described KELnull allele on a KEL*01 backbone and 184Tdel. In the 121 KEL:1,-2 individuals, nine (7.4%) were found to display a discordant KEL:1,-2 phenotype and KEL*01/KEL*02 genotype. Three novel silent KEL*02 alleles were described: 1084C>A, 1708G>A, and IVS11+5g>a. CONCLUSION: The number of silent KEL alleles and the notion that KEL null alleles are on a KEL*02 background may evolve in the coming years. Systematic DNA analysis showed that the number of discordant phenotype/genotype results, related to silent KEL*02 alleles was higher than expected in France. These data emphasize that clinical practice based on DNA analysis for blood group antigens requires caution and should improve the performance of the blood group phenotype prediction.

RHD*DOL1 and RHD*DOL2 encode a partial D antigen and are in cis with the rare RHCE*ceBI allele in people of African descent.

BACKGROUND: Several studies showed in people of African descent the existence of a genetic linkage between RHD alleles encoding a variant D antigen and a given altered RHCE*ce allele. RHCE*ceBI is a rare allele encountered in people of African descent, that encodes a Hr- hr(S) - Rhce protein. Our study shows that RHCE*ceBI appears to be genetically linked to two very similar variant RHD alleles, RHD*DOL1 and RHD*DOL2, and demonstrates for the first time that DOL-2 is a partial D antigen. STUDY DESIGN AND METHODS: After finding out an individual with both RHCE*ceBI and RHD*DOL presumed to be in cis, we hypothesized a genetic linkage between those two genes. All individuals (n = 7) known to carry RHCE*ceBI in our laboratory, including the index case, were fully investigated at the serologic and molecular level. RESULTS: One individual with alloanti-D, being homozygous for RHCE*ceBI and RHD*DOL2, allowed us to confirm the genetic linkage between those two genes, as well as the partial D status of DOL-2. In the six RHCE*ceBI remaining individuals, three were found with RHD*DOL2 and 3 with RHD*DOL1, likely in cis. Three of them made an alloanti-D; one was DOL-1 and two were DOL-2. CONCLUSION: The rare RHCE*ceBI allele appears to be in cis either with RHD*DOL1 or with RHD*DOL2 in people of African descent. DOL-1 and DOL-2 must be considered as partial D antigens. We recommend a systematic search for RHD*DOL1 and RHD*DOL2 in people found to carry RHCE*ceBI and vice versa, especially in patients with sickle cell disease.

Molecular analysis of patients with weak D and serologic analysis of those with anti-D (excluding type 1 and type 2).

Whether or not patients whose red blood cells (RBCs) carry certain weak D types produce anti-D, and if they do whether it is allo- or auto anti-D, remains controversial. The aim of this study was to determine the serologic features of anti-D in individuals expressing a weak D other than type 1 or type 2 and to assess whether the anti-D was an allo- or autoantibody. Serologic D typing and molecular analyses were performed on 748 individuals.Serologic characterization of anti-D included autologous controls,direct antiglobulin test, elution, and titration of anti-D before and after adsorption of serum onto autologous RBCs. From molecular analyses, 459 individuals exhibited a weak D type. We described seven novel RHD variant alleles. The most frequent types of weak D were type 1 (30.1%), type 2 (23.7%), type 4.0 (10.2%), type 4.2.2(20.3%), type 11 (3.9%), and type 15 (3.7%). Anti-D was identified in the sera of 9 of 47 individuals with weak D type 4.0, in 14 of 93 with weak D type 4.2.2, in 1 of 18 with weak D type 11, in 1 of 17 with weak D type 15, and in 1 weak D type 33 individual.Anti-D was demonstrated to be an alloantibody in weak D type 4.0, type 4.2.2, and type 15 individuals, but an autoantibody in weak D type 11 and type 33 individuals. In conclusion, only a complete serologic investigation of individuals with a given weak D type identified by molecular analysis allows concluding on the nature of the antibody. Transfusing weak D type 4.2.2 and type 15 patients with D- RBC units and proposing anti-D immunoprophylaxis to women with these weak D types should be considered.

Characterization of the novel HLA-A*33:220 allele by next-generation sequencing.

HLA-A*33:220 differs from HLA-A*33:03:01:01 by one nucleotide substitution in codon 245 in exon 4.