Molecular Diagnostics on Smartphone

During the COVID-19 pandemic, point-of-care genetic testing (POCT) devices were used for on-time and on-site detection of the virus, which helped to prevent and control the spread of the pandemic. Smartphones, which are widely used electronic devices with many functions, have the potential to be used as a molecular diagnostic platform for universal healthcare monitoring. Several integrated diagnostics platforms for the real-time and end-point detection of COVID-19 were developed using the functions of smartphones, such as the operating system, power, sound, camera, data storage, and display. These platforms use the 5V output power of smartphones, which can be amplified to power a micro-capillary electrophoresis system or a thin-film heater, and the CMOS camera of smartphones can capture the color change during a colorimetric loop-mediated isothermal amplification test and detect fluorescence signals. Smartphones can also be used with self-written web-based apps to enable automatic and remote pathogen analysis on POCT platforms. Our lab developed a handheld micro-capillary electrophoresis device for end-point detection of SARS-CoV-2, as well as an integrated smartphone-based genetic analyzer for the qualitative and quantitative colorimetric detection of foodborne pathogens with the help of a custom mobile app. © 2023 SPIE.

Gene Editing/Gene Therapies: BIOREACTOR RECOMBINANT PRODUCTION OF SARS-COV-2 VIRUS ANTIGENS IN CHO CELL CLONES BY USING NEW UCOE LENTIVIRAL VECTOR SYSTEM

Background & Aim: The new UCOE models we have recently developed, tested on many cell groups (including mouse ES and human iPS cells) and human mAb recombinant production studies as well, shows a powerful resistance to DNA methylation- mediated silencing and provides a higher and stable transfection profile. By the urgent need of vaccine development for COVID-19 during the pandemic, in this study we aimed to produce a potential recombinant vaccine by using the new generation UCOEs models of our own design. Methods, Results & Conclusion(s): Existing new-generation UCOE models and standard plasmid vectors to be used as control group were provided. Then, the sequences related to the PCR method were amplified for sufficient stock generation and cloning experiments. Verification in the plasmid vector was carried out in gel electrophoresis. Transfection of 293T cells was performed with clone plasmids carrying antigen genes and plasmids carrying genetic information of lentivirus units for the production of lentiviral vectors. Afterwards, 293T cells produced lentiviral vectors carrying antigen genes. Harvesting of these vectors was carried out during 48th and 72nd hours. Afterwards, CHO cells were transduced with appropriate quantity of lentiviral vectors. Isolation and purification of targeted proteins from the relevant medium were performed by HPLC and Q-TOF methods. A part of the spike and nucleocapsid gene sequences of COVID-19 were firstly cloned into our UCOE models. These UCOEs plasmids were then transferred into 293T cells along with plasmids carrying the genes that will form the lentivirus vectors (LVs). After harvesting and calculation of LV vector titers, the cloned vectors were then transfected into the CHO cells which the targeted recombinant production of the antigen proteins will be carried out. Antigenic structures were then isolated from the culture medium of CHO cells in following days for confirmation. Using HPLC and qTOF mass spectrometer methods, these structures in the medium were confirmed to be the units of spike and nucleocapsid proteins of the COVID-19 virus. In order to produce large amount of the recombinant antigens, the culture was then carried out with bioreactors in liters. At the final stage, these recombinantly produced antigen proteins were tested on rats to measure their immunogenic responses, and the study recently been completed successfully as a potential recombinant vaccine against COVID-19.Copyright © 2023 International Society for Cell & Gene Therapy

Phenotypic Characteristics and Clonal Relationships of Stenotrophomonas maltophilia Isolates in Hospitalized Adults from a Private Center in Lima, Peru.

Stenotrophomonas maltophilia is an opportunistic pathogen, often associated with nosocomial infections. Ten S. maltophilia were isolated from clinical samples during the period January 2021 and June 2022. Eight (80%) patients had cancer as a background disease and 2 patients had coronavirus disease 2019. A fatal outcome was recorded in 4 cases (40% of patients). All the isolates were susceptible to minocycline and levofloxacin. Trimethoprim/sulfamethoxazole and ceftazidime resistance rates were 20% and 40% respectively. Eight different patterns were observed by Pulsed-Field Gel Electrophoresis, only two isolates being clonally identical. The isolation of S. maltophilia in clinical settings requires the implementation of infection prevention measures.

Advancements in portable instruments based on affinity-capture-migration and affinity-capture-separation for use in clinical testing and life science applications.

The shift from testing at centralized diagnostic laboratories to remote locations is being driven by the development of point-of-care (POC) instruments and represents a transformative moment in medicine. POC instruments address the need for rapid results that can inform faster therapeutic decisions and interventions. These instruments are especially valuable in the field, such as in an ambulance, or in remote and rural locations. The development of telehealth, enabled by advancements in digital technologies like smartphones and cloud computing, is also aiding in this evolution, allowing medical professionals to provide care remotely, potentially reducing healthcare costs and improving patient longevity. One notable POC device is the lateral flow immunoassay (LFIA), which played a major role in addressing the COVID-19 pandemic due to its ease of use, rapid analysis time, and low cost. However, LFIA tests exhibit relatively low analytical sensitivity and provide semi-quantitative information, indicating either a positive, negative, or inconclusive result, which can be attributed to its one-dimensional format. Immunoaffinity capillary electrophoresis (IACE), on the other hand, offers a two-dimensional format that includes an affinity-capture step of one or more matrix constituents followed by release and electrophoretic separation. The method provides greater analytical sensitivity, and quantitative information, thereby reducing the rate of false positives, false negatives, and inconclusive results. Combining LFIA and IACE technologies can thus provide an effective and economical solution for screening, confirming results, and monitoring patient progress, representing a key strategy in advancing diagnostics in healthcare.

Production of Sars-Cov-2 Membrane Protein as Better Target Protein for Diagnostic Application

Intro: The COVID-19 pandemic is caused by the SARS-CoV-2 virus, an enveloped RNA of the coronavirus family. The advancement in molecular technology and biochemistry has accelerated the development of diagnostic reagents and assays. Much attention has been focused on the S protein, but the high mutation rate in this region could lead to false negative results. Thus, a better target protein for diagnostic application is needed for accurate detection. Method(s): Nucleotide sequences encoded for membrane (M) glycoprotein gene region of SARS-CoV-2 from Malaysian isolates were extracted from GISAID, aligned, and selected accordingly. The DNA plasmid was commercially synthesized with codon optimization for Escherichia coli (E. coli), and the presence of the M gene was confirmed by PCR. The plasmid was then transformed into E. coli. Later, the expression of M glycoprotein was induced, separated on an SDS-PAGE gel, and transferred onto a nitrocellulose membrane, followed by immunostaining. Finding(s): The analysis of the M glycoprotein against the Omicron strains demonstrated that the amino acid is conserved (99.5%). The M glycoprotein was successfully expressed and detected with antibodies from SARS-CoV-2 infected patients at ~26 kDa. The protein is currently upscale for the generation of monoclonal Ab (Mab). Discussion(s): The M protein of SARS-CoV-2 is more conserved among the virus and also has been reported to confer antigenic properties. Selection of M protein perhaps a better option compared to current detection assays that use spike (S) protein, which could lead to false negative results, as this gene region particularly the ribosome-binding domain (RBD) rapidly undergoes mutations. The utilization of M protein potentially improves negative predictive value (NPV) of the diagnostic test. Conclusion(s): Further development of diagnostic reagents is needed to improve the assay's specificity. The newly developed M protein and the MAb can be used to generate a more accurate viral detection assay.Copyright © 2023

Hiv Detection in Wastewater as a New Epidemiological Tool

Background: Wastewater represents a broad, immediate, and unbiased accounting of the pathgens in the population. We aimed to develop methods to track HIV in wastewater utilizing a viral detection pipeline adapted from platforms developed to track SARS-COV-2. Method(s): We used samples from 6 wastewater treatment plants in the Houston area. We focused on regions of higher prevalence and lower prevalence. First, employing wastewater processing and nucleic acid extraction methods described by our group to detect SARS-COV-2, we tested a single high and low prevalence site in triplicate with all 3 primer sets. nucleic acid extracts from HIV and SIV cell culture supernatants were used as controls. Next, in subsequent samples, RT-PCR reactions with detections were subjected to gel electrophoresis to determine the amplified product sizes. To further confirm HIV detection, we sequenced the RT-PCR products and compared the proportion of reads which mapped to the expected amplified product. In a later set of studies, we fractionated samples into supernatant and pellet. We further tested HIV presence by performing whole virome sequencing on the extracts from some samples that produced detections and mapped reads to published genomes. A crAssphage genome was used as a negative control. Result(s): Samples from all sites resulted in signal detection at least once. Only reactions with gag and pol primers appeared to amplify the expected product. Products from the HIV positive control mapped almost exclusively to the HIV genome (97-100% of reads), with a fraction of reads from the SIV negative control doing the same (16-18% of reads). The ltr and pol products did not map the HIV genome while gag products did (34-44% of reads). Among the fractionated sample, in total, 6 supernatant fractions produced no detection compared to 7 of 8 pellet fractions. The whole virome sequencing produced reads that mapped to the HIV genome with at least 8X depth coverage. The sample with the lowest Ct detection (26) yielded HIV coverage several logs greater than those samples with higher Ct detection (37). Reads from all samples mapped to at least 20% of the HIV genome. Conclusion(s): This work provides the first evidence that HIV can be detected in municipal wastewater systems and has the potential to be developed into a new public health tool.

Isolation and Characterization of Protein Complexes of Overexpressed Human Serum Amyloid A from Cytokines-Induced Hepg2 Cells

Serum Amyloid A (SAA) is an apolipoprotein found in the serum of many vertebrate species and is associated with the acute-phase reaction in the body with expression levels reaching up to a 1000-fold increase. The loss of its alpha-helix conformation during its expression peak is directly linked to secondary amyloidosis. Recent studies have been suggested to play a role in cholesterol and HDL metabolism, retinol transport and tumor pathogenesis. Moreover, high SAA concentration in blood have been correlated with severe symptoms or death in patients with COVID-19. However, how this protein is involved in so many diseases is uncertain or not completely understood. Therefore, the purpose of this research is to determine which protein-protein interactions with SAA occur in human cells, and to predict its biochemical role based on new discovered complexes. Two major isoforms overexpressed during an acutephase reaction, human SAA1 and SAA2, are the focus of this study. Both are primarily produced in hepatocytes. HepG2 cells were cultured and induced with interleukin-1b, interleukin-6, LPS and retinol. Protein complexes associated with SAA will be isolated through a co-Immunoprecipitation technique, resolved by SDS-PAGE, and characterized by mass spectrometry. Our hypothesis focus on those protein complexes with SAA to explain how this protein lead other undiscovered metabolic pathways involved in both cellular and survival regulation. Special thanks to The Science and Technology Competency & Education Core (Stce) for Undergraduate and Graduate Junior Research Associates Working Program from the Puerto Rico IDeA Network Biomedical Research Excellence for funding part of this research.Copyright © 2023 The American Society for Biochemistry and Molecular Biology, Inc.

Plasma markers of Covid-19 severity: A pilot study

SARS-CoV-2 infected patients show heterogeneous clinical presentations ranging from mild symptoms to severe respiratory failure and death. Consequently, various markers reflect certain disease presentations. Our cohort included moderate (n = 10) and severe (n = 10) COVID-19 patients, and 10 healthy controls. We determined plasma levels of nine acute phase proteins by nephelometry, full-length (M65), caspase-cleaved (M30) cytokeratin 18, and ADAMTS13 (a disintegrin-like and metalloprotease with thrombospondin type-1 motif 13) by ELISA. In addition, we examined whole plasma N-glycosylation by capillary gel electrophoresis coupled to laser-induced fluorescence detection. When compared to healthy controls, COVID-19 patients had significantly lower concentrations of ADAMTS13 and albumin (ALB) but higher M30, M65, alpha-1-acid glycoprotein, alpha1-antitrypsin (AAT), ceruloplasmin, haptoglobin, and highsensitivity C-reactive protein. The concentrations of alpha1-antichymotrypsin, alpha2-macroglobulin and serum amyloid A proteins did not differ. We found significantly higher levels of AAT and M65 but lower ALB in severe compared to moderate COVID-19 patients. N-glycan analysis of the serum proteome revealed increased levels of oligomannose and sialylated di-antennary glycans, while the non-sialylated di-antennary glycan A2G2 significantly decreased in COVID-19 patients compared to controls. COVID-19-associated changes in levels and N-glycosylation of specific plasma proteins highlight involvement of different pathophysiological mechanisms and grant further investigations.

Erythema nodosum -Expanding diagnostic workout in COVID-19 pandemic

Case report Erythema nodosum (EN) is considered a delayed type IV hypersensitivity reaction, triggered by exposure to an antigen, which diagnostic workout is usually challenging. Several conditions have been described as possible causes for EN, including infections, sarcoidosis, pregnancy, neoplasic and inflammatory diseases. Rarely, vaccines such as tetanus, diphtheria, BCG, hepatitis B, human papillomavirus, malaria, rabies, smallpox, typhoid, and cholera have been associated with subsequent EN. We present a 31-year- old leucodermic female with suppurative adenitis, who developed painful erythematous nodules on the pretibial area of the lower limbs. Ten days prior to presentation she had received the first dose of the COVID-19 mRNA-1273 vaccine. Fever, lymphadenopathy, fatigue, weight loss, arthritis, cough, diarrhoea, other organ-specifc symptoms and close contact with tuberculosis were excluded. She was under oral contraception for several years, that was not discontinued. Pregnancy was excluded. No positive signs were detected on physical examination besides the referred nodules. Laboratory tests revealed a normal complete blood count, erythrocyte sedimentation rate, C-reactive protein, antistreptolysin O titer, renal and hepatic tests. Interferon-gamma release assay was negative. Circulating rheumatoid factor was normal, anti-nuclear, anti-double stranded DNA and anti-neutrophil cytoplasmatic antibodies were negative. Angiotensin converting enzyme and protein electrophoresis were normal. Hepatitis B and C, HIV 1/2 and syphilis serologic profiles were negative. Urinalysis and fecal calprotectin were unremarkable. The patient was treated with naproxen and topic betamethasone dipropionate. Violaceous involution was reported, with complete resolution of the EN lesions over the following month. In the literature, there are rare reports of EN following SARS-COV2 infection and also after COVID-19 vaccination. To our knowledge this is the second report of EN after the COVID-19 mRNA-1273 vaccine. This case highlights the importance of clinical awareness for the possible association of COVID-19 vaccination and EN, adding to the already extensive list of causes included in the etiological investigation of these patients.

Abnormal echocardiographic findings after COVID-19 infection: A multicenter registry

Background: The Coronavirus Disease 2019 (COVID-19) pandemic has transformed health systems worldwide. There is conflicting data regarding the degree of cardiovascular involvement following infection, generating uncertainty in patients and an additional healthcare burden with increased diagnostic testing. A registry was designed to evaluate the prevalence of echocardiographic abnormalities in Latin American adults recovered from COVID-19. Method(s): We prospectively evaluated 595 participants (mean age 45.5+/-14.9 years;50.8% female) from 10 institutions in Argentina and Brazil. Echocardiographic studies were conducted with General Electric equipment;2DE imaging and global longitudinal strain (GLS) of both ventricles were performed. Comparisons between groups were made with Chisquare, Fisher and Student's t-test. Logistic regression was performed to determine variables associated with abnormal echocardiogram findings. Result(s): A total of 61.7% of the participants denied any relevant cardiovascular medical history. Table 1 summarizes the comorbidities of the included patients. The majority of patients (82.5%) had the disease at home or in an out-of-hospital center. Of the patients who required hospitalization, 15.3% were in a general ward, 1.9% in intensive care and 0.3% required mechanical ventilation during the disease. The median time between infection and performance of the echocardiographic study was two months (IQR 1- 3 months). Among patients who reported symptoms following COVID-19 recovery (41.8%), the most frequently reported was dyspnea (47.4%), followed by mild symptoms such as asthenia, arterial hypertension or palpitations (32.9%), 12.9% referred chest pain, 6% of patients reported dyspnea and chest pain, and 0.8% reported various other symptoms. The mean left ventricular ejection fraction (LVEF) was 61.0+/-5.5% and the mean left atrial volume was 33.1+/-13.2 ml/m2. In patients without prior comorbidities, 8.2% had some echocardiographic abnormality (Figure 1). We found no significant differences in LVEF between symptomatic and asymptomatic patients (61.4% versus 60.6% respectively, p=0.104). Symptomatic patients showed slightly reduced GLS (-20.3% versus -20.9%, p=0.012) with a trend in the same direction in the RV free wall GLS (-25.6% versus -26.3%, p=0.103). Male patients were more likely to have any new echocardiographic abnormalities (OR 2.82, p=0.002). Time elapsed since infection resolution (p=0.245), the presence of symptoms (p=0.927), or history of hospitalization during infection (p=0.671) did not have any correlation with echocardiographic abnormalities. The difference between sexes remains unchanged after adjusting for left atrial volume, wall thicknesses, diastolic function and abnormal wall motion. Conclusion(s): Our results suggest that cardiovascular abnormalities after COVID-19 infection are rare and usually mild, especially in cases of mild disease. These abnormalities may be more frequent among males.

New onset chronic spontaneous urticaria post COVID-19 vaccination in South Africa

Background: Chronic spontaneous urticaria (CSU) is a common chronic inflammatory disease. There have been small case series of new onset CSU post COVID-19 infection as well as reports of new onset CSU or worsening of existing CSU post COVID-19 vaccination. Dermatological side effects post COVID-19 vaccine are typically delayed, self-limiting urticaria. We have described the characteristics of patients who have developed new onset CSU post COVID-19 vaccination. Method(s): All patients referred to the UCT Lung Institute Allergy clinic from the initiation of the COVID-19 vaccine roll out (February 2021) were reviewed to identify patients that developed new onset CSU within 12 weeks of receiving a COVID-19 vaccine. Medical history, response to therapy, and available laboratory investigations were reviewed by clinic physicians. Result(s): We identified seven patients that developed CSU post COVID-19 vaccine. The median age of the cohort is 39 (IQR 32-45) and the majority are female (n = 5). The most common vaccine was the Pfizer-BioNTech BNT162b2 mRNA COVID-19 vaccine (n = 6, 85.7%), and one patient received the Jansen Ad26.COV2.S vaccine. No patients had COVID-19 infection prior to vaccination and only one patient contracted COVID-19 post vaccination. The median time to the development of symptoms post vaccination was 14 days (IQR 2;44) and the median time to diagnosis was 90 days (IQR 45;120). Most patients (n = 4) reported angioedema and urticaria, one patient reported isolated angioedema, and two isolated urticaria. The median initial UAS7 score was 37.5 (IQR 24.5;46) and the initial CU-Q2oL score was 72 (IQR 56;76) indicating severe disease activity. All but one patient had a history of atopy with the most common diagnoses being allergic rhinitis (n = 5) and atopic dermatitis (n = 3). All patients had normal eosinophil counts and over half of the patients (n = 4) had an elevated total IgE level (median 26.4 [IQR 9.8;194]). All patients were HIV negative and one patient had positive Helicobacter pylori serology. All had normal serum protein electrophoresis, thyroid function (with negative thyroid autoantibodies), and negative antinuclear antibodies. All patients started on high dose antihistamine therapy with 71.1% having partial or no response to therapy. Conclusion(s): New onset CSU is a rare side effect of COVID-19 vaccination with poor response to high dose antihistamine therapy. It is important that allergists and physicians are aware of the possibility of new onset CSU post COVID-19 vaccine and further research is needed to identify risk factors.

A Microfluidic Biosensor for Rapid Detection of Covid-19

We have designed, fabricated, and tested a MEMS-based impedance biosensor for accurate and rapid detection of severe acute respiratory syndrome coronavirus 2 (SARS-COV-2) using of clinical samples. The device consists of focusing region that concentrate low quantities of the virus present in the samples to a detectable threshold, trap region hat maximize the captured virus, and detection region to detect the virus with high selectivity and sensitivity, using an array of interdigitated electrodes (IDE) coated with a specific antibody. Changes in the impedance value due to the binding of the SARS-COV-2 antigen to the antibody will indicate positive or negative result. The device was able to detect inactivated SARS-COV-2 antigen present in phosphate buffer saline (PBS) with a concentration as low as 50 TCID50/ml in 30 minutes. In addition, the biosensor was able to detect SARS-COV-2 in clinical samples (swabs) with a sensitivity of 84 TCID50/ml, also in 30 minutes. © 2023 IEEE.

A contrasting diagnosis: iododerma

An 86-year-old woman presented to the emergency department with acute shortness of breath. She was treated with intravenous furosemide for acute-on-chronic heart failure. Her past medical history included atrial fibrillation, hypertension, diverticulosis and hypothyroidism. Rivaroxaban and levothyroxine were her only long-term medications. On day 5 of hospital admission, she developed painful haemorrhagic and purulent bullae on her dorsal hands, head and neck. These evolved to large suppurative, vegetative plaques over a 72 h period and she developed additional lesions on her trunk, upper back and thighs. The patient had routine blood tests, which showed a raised C-reactive protein at 260 mg L-1, and an acute kidney injury with a glomerular filtration rate of 54 mL-1 min-1. She had a negative COVID-19 swab, and swabs from the lesions for bacterial culture and viral polymerase chain reaction were negative. She had a normal serum protein electrophoresis, immunoglobulin, antinuclear antibody and antineutrophil cytoplasmic antibody. She had computed tomography of her chest 24 h prior to the onset of her lesions, which showed mild bilateral pleural effusions in keeping with fluid overload secondary to heart failure. A biopsy taken from her hand showed orthokeratosis and parakeratosis, and there was bulla formation subepidermally. There was a dense neutrophilic infiltrate with microabscess formation with scattered eosinophils and lymphocytes. There was no evidence of vasculitis. Direct immunofluorescence was negative and a tissue culture for atypical mycobacteria was negative. The patient was commenced on high-dose intravenous methylprednisolone at 500 mg for 3 days followed by 40 mg prednisolone orally for 1 week, but there was a limited response. Our initial differential was Sweet syndrome or pyoderma vegetans;however, the patient had no fevers and no risk factors (malignancy, inflammatory disease, infection, etc.). She also had no response to high-dose oral prednisolone. Given the timing of her CT examination in relation to her acute dermatosis and the use of radioiodine for contrast, we assessed the patient's serum iodine and urine iodine. These were both high at 1.02 mmol L-1 (reference interval 0.32- 0.63) and 3.46 mmol L-1 (reference interval 0.0-2.43), respectively. A diagnosis of iododerma was made. The patient's eruption slowly resolved and at 12 weeks there was evidence of postinflammatory skin changes only. Her urine and serum iodine were rechecked, and both had normalized. In the last 20 years there have been approximately 20 case reports of iododerma. Most have been following iodine contrast use in patients with abnormal kidney function, like our patient. Most describe an acneiform eruption that subsequently evolves to vegetative plaques (Chalela JG, Aguilar L. Iododerma from contrast material. N Engl J Med 2016;374: 2477). Iododerma is largely a diagnosis of exclusion, but histopathology and urine and serum iodine levels can help support diagnosis.

Protease Activity in the Excretory-Secretory Products of Nymphal Stage of Linguatula Serrata: First Detection of a Metalloprotease

The aim of this study was to evaluate the presence of proteases and determine the main protease present in the excretory-secretory products (ESPs) from nymphal stage of Linguatula serrata. Infected mesenteric lymph nodes of goats were collected from Tabriz slaughterhouse, northwestern Iran. Recovered Linguatula serrata nymphs were immersed in culture medium (MEM), then ESPs were collected and protease activity in presence of specific inhibitors was assayed. Protease enzyme was fur-ther characterised by SDS-PAGE. The results of this study showed that the main protease in the ESPs from the nymphal stage of L. serrata was a metalloprotease that was resistant to heat. In conclusion, these data show that a major protease secreted by the larval stage of L. serrata exhibited properties that may play a role in the pathogenesis of L. serrata nymphs.Copyright © 2023, Trakia University. All rights reserved.

New-Onset Chronic Spontaneous Urticaria Post Covid-19 Vaccination in South Africa

INTRODUCTION Chronic spontaneous urticaria (CSU) is a common chronic inflammatory disease. There have been small case series of new onset CSU post-COVID-19 infection and reports of new onset or worsening of existing CSU post COVID-19 vaccination. A dermatological side-effect post COVID-19 vaccine is typically delayed, self-limiting urticaria. We have described the characteristics of patients who have developed new-onset CSU post COVID-19 vaccination. METHOD All patients referred to the Allergy Clinic since the initiation of the COVID-19 vaccine roll-out in South Africa were reviewed to identify patients who had developed new-onset CSU within 12 weeks of receiving a COVID-19 vaccine. Medical history, response to therapy and available laboratory investigations were reviewed by clinic physicians. RESULTS We identified seven patients who developed CSU post COVID-19 vaccination. The median age of the cohort is 39 (IQR 32-45) and the majority are female (n = 5). The most common vaccine was the Pfizer-BioNTech BNT162b2 mRNA COVID-19 vaccine (n = 6;85). The median time to the development of symptoms post-vaccination was 14 days (IQR 2;44) and the median time to diagnosis was 90 days (IQR 45;120). The median initial UAS7 score was 37.5 (IQR 24.5;46) and the initial CU-Q2oL score was 72 (IQR 56;76), which indicated severe disease activity. All but one patient had a history of atopy, with the most common diagnoses being allergic rhinitis (AR) (n = 5) and atopic dermatitis (AD) (n = 3). All the patients had normal eosinophil counts and more than half of the patients (n = 4) had an elevated total IgE level (median 26.4 [IQR 9.8;194]). All of the patients were HIV-negative. All of them had normal serum protein electrophoresis, thyroid function (with negative thyroid autoantibodies) and negative antinuclear antibodies. All of them started on high-dose antihistamine therapy, with 71.1% having partial or no response to the therapy. CONCLUSION New-onset CSU is a rare side-effect of COVID-19 vaccination, with poor response to high-dose antihistamine therapy. It is important that allergists and physicians are aware of the possibility of new-onset CSU post COVID-19 vaccination and further research is needed to identify any risk factors.

Balance between alpha-1Antitrypsin and Human Neutrophil Elastase (a1AT/HNE) in Bronchoalveolar Lavage fluid (BALf) of severe COVID19 patients

Neutrophils (Neu) play a pathogenic role in COVID19 by releasing Neutrophils Extracellular Traps (NETs) or HNE. Being HNE inhibited by a1AT, supplementation of this protein has been proposed. We aim to study a1AT/HNE balance in BALf from ICU admitted COVID19 patients. To assess HNE, a1AT and HNE/a1AT complexes, 33 COVID 19 BALf samples were analysed by means of ELISA or gel-Electrophoresis + Western Blot. Proteins bound to a1AT or HNE were identified by Liquid chromatography-mass spectrometry. NETs release (PMA stimulated Neu +/- a1AT) was analysed by confocal microscopy. Both HNE and a1AT were clearly detectable in BALf at high levels. Contrary to what previously observed in other settings (Bronchiolits obliterans) (Cagnone, M. et al. High Throughput 2019;8(1):5) we couldn't detect any HNE/ a1AT complex in COVID19 even when purified HNE was added to samples (Fig 1a). HNE was found to be bound to acute phase proteins, histones and C3. Due to the relevant role of NETs, we assessed the ability of free a1AT to bind to histones. Although this binding was confirmed, a1AT wasn't able to inhibit NETs formation (Fig 1b). Despite the finding of a high burden of free and bound HNE in COVID 19 BALf, the formation of HNE/ a1AT inhibitory complex is prevented. Furthermore, a1AT binds to histones but does not prevent NETs formation and their noxious activity.

Tixagevimab Plus Cilgavimab Does Not Affect the Interpretation of Electrophoretic and Free Light Chain Assays.

OBJECTIVES: There is concern that the anti-severe acute respiratory syndrome coronavirus 2 therapeutic monoclonal antibodies, used as preexposure prophylaxis in patients with multiple myeloma, may appear as a detectable monoclonal protein by electrophoretic methods, resulting in misinterpretation or inability to measure therapeutic responses in some patients. In this pilot study, we characterize the effect of tixagevimab plus cilgavimab (Evusheld; T + C) on interpretation of serum protein electrophoresis (SPE), immunofixation electrophoresis (IFE), and serum free light chain (sFLC) assays. METHODS: We performed spiking experiments with T + C at serum maximum concentration following a 300-mg dose (1× Cmax) and at 10 times the concentration of Cmax (10× Cmax) with pooled serum samples. SPE and IFE technical procedures were performed on the SPIFE 3000, and sFLC and immunoglobulin G1 (IgG1) subtype quantitation was performed on the Optilite. RESULTS: T + C-associated interference was not visible as an M-spike in normogammaglobulinemic pooled samples. Hypogammaglobulemic pooled samples at 10× Cmax demonstrated an M-spike in SPE and immunoglobulin Gκ pattern in IFE. No increases were noted in the results of sFLC or IgG1 levels. CONCLUSIONS: This study indicates that T + C at pharmacologic Cmax is unlikely to interfere with SPE, IFE, sFLC, or IgG1 analyses when spiked into patient serum samples, but further evaluation of recently injected patients may be warranted.

Present status of microfluidic PCR chip in nucleic acid detection and future perspective

Polymerase chain reaction (PCR) amplifies specific fragment of DNA molecules and has been extensively applied in fields of pathogens and gene mutation detection, food safety and clinical diagnosis which on the other hand, holds the drawbacks of large size instrument, high heat dissipation etc. It has been demonstrated that microfluidics technique coupling with PCR reaction exhibits characteristics of integration, automatization, miniaturization, and portability. Meanwhile, various designed fabrication of microchip could contribute to diverse applications. In this review, we summarized major works about a variety of microfluidic chips equipped with several kinds of PCR techniques (PCR, RT-PCR, mPCR, dPCR) and detection methods like fluorescence, electrochemistry, and electrophoresis detection. The development and application of PCR-based microfluidic chip in pathogen and gene mutation detection, diseases prevention and diagnosis, DNA hybridization and low-volume sample treatment were also discussed. Copyright © 2022 Elsevier B.V.

Identification of SARS-CoV-2 Main Protease (Mpro) Cleavage Sites Using Two-Dimensional Electrophoresis and In Silico Cleavage Site Prediction.

The main protease (Mpro) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) plays a crucial role in its life cycle. The Mpro-mediated limited proteolysis of the viral polyproteins is necessary for the replication of the virus, and cleavage of the host proteins of the infected cells may also contribute to viral pathogenesis, such as evading the immune responses or triggering cell toxicity. Therefore, the identification of host substrates of the viral protease is of special interest. To identify cleavage sites in cellular substrates of SARS-CoV-2 Mpro, we determined changes in the HEK293T cellular proteome upon expression of the Mpro using two-dimensional gel electrophoresis. The candidate cellular substrates of Mpro were identified by mass spectrometry, and then potential cleavage sites were predicted in silico using NetCorona 1.0 and 3CLP web servers. The existence of the predicted cleavage sites was investigated by in vitro cleavage reactions using recombinant protein substrates containing the candidate target sequences, followed by the determination of cleavage positions using mass spectrometry. Unknown and previously described SARS-CoV-2 Mpro cleavage sites and cellular substrates were also identified. Identification of target sequences is important to understand the specificity of the enzyme, as well as aiding the improvement and development of computational methods for cleavage site prediction.