SARS-CoV-2 infection disrupts trophoblast function and placental syncytial barrier integrity via its accessory protein, ORF3a

Problem: Although it is rare for a SARS-CoV-2 infection to transmit vertically to the fetus during pregnancy, there is a significantly increased risk of adverse pregnancy outcomes due to maternalCOVID- 19. However, there is a poor understanding of such risks because mechanistic studies on how SARS-CoV-2 infection disrupts placental homeostasis are significantly lacking. The SARS-CoV-2 proteome includes multiple structural and non-structural proteins, including the non-structural accessory proteinORF3a. The roles of these proteins in mediating placental infection remain undefined. We and others have shown that autophagy activity in placental syncytium is essential for barrier function and integrity. Here, we have used clinical samples and cultured trophoblast cells to evaluate syncytial integrity of placenta exposed to SARS-CoV-2. The objective of our study was to investigate potential mechanisms through which SARS-CoV-2 impairs placental homeostasis and causes adverse pregnancy outcomes. We tested the central hypothesis that an essential SARS-CoV-2 non-structural and accessory protein, ORF3a, uniquely (amongst multiple viral proteins tested) with a novel three-dimensional structure andwith no homology to any other proteins is a key modulator of placental trophoblast cell dynamics via autophagy and intracellular trafficking of a tight junction protein (TJP), ZO-1. Method(s): We used clinical samples and cultured trophoblast cells to evaluate syncytial integrity of placentas exposed to SARS-CoV- 2. Autophagic flux was measured in placental villous biopsies from SARS-CoV-2-exposed and unexposed pregnant women by quantifying the expression of autophagy markers, LC3 and P62. Trophoblast cells (JEG-3, Forskolin-treated JEG-3, HTR8/SVneo, or primary human trophoblasts (PHTs)) were transfected with expression plasmids encoding SARS-CoV-2 proteins including ORF3a. Using western blotting, multi-label immunofluorescence, and confocal imaging, we analyzed the effect of ORF3a on the autophagy, differentiation, invasion, and intracellular trafficking of ZO-1 in trophoblasts. Using coimmunoprecipitation assays, we tested ORF3a interactions with host proteins. t-tests and one-way analyses of variance (ANOVAs) with post hoc tests were used to assess the data, with significance set at P < .05. Result(s): We discovered :1) increased activation of autophagy, but incomplete processing of autophagosome-lysosomal degradation;2) accumulation of protein aggregates in placentas exposed to SARS-CoV- 2. Mechanistically, we showed that the SARS-CoV-2 ORF3a protein, uniquely 3) blocks the autophagy-lysosomal degradation process;4) inhibits maturation of cytotrophoblasts into syncytiotrophoblasts (STBs);5) reduces production ofHCG-beta, a key pregnancy hormone that is also essential for STB maturation;and 6) inhibits trophoblast invasive capacity. Furthermore, ORF3a harbors an intrinsically disordered C-terminus withPDZ-bindingmotifs.We show for the first time that, 7) ORF3a binds to and co-localizes with the PDZ domain of ZO-1, a junctional protein that is essential for STB maturation and the integrity of the placental barrier. Conclusion(s): Our work outlines a new molecular and cellular mechanism involving the SARS-CoV-2 accessory protein ORF3a that may drive the virus's ability to infect the placenta and damage placental syncytial integrity. This implies that the mechanisms facilitating viral maturation, such as the interaction of ORF3a with host factors, can be investigated for additional functionality and even targeted for developing new intervention strategies for treatment or prevention of SARS-CoV-2 infection at the maternal-fetal interface.

First isolation and characterization of the prototype strain of the SARSCoV-2 virus at the beginning of the COVID-19 pandemic in Peru

Introduction: Currently, isolated from SARS-CoV-2 virus exceed 600 million cases in the world. Objective(s): Isolation and characterization of the SARS-CoV-2 virus causing COVID-19 at the beginning of the pandemic in Peru. Method(s): Twenty nasal and pharyngeal swab samples were isolated from SARS-CoV-2 using two cell lines, Vero ATCC CCL-81 and Vero E-6;virus identification was performed by RT-PCR and the onset of cytopathic effect (CPE) was evaluated by indirect immunofluorescence and subsequent identification by genomic sequencing. One of the most widely circulating isolates were selected and named the prototype strain (PE/B.1.1/28549/2020). Then 10 successive passages were performed on Vero ATCC CCL-81 cells to assess mutation dynamics. Result(s): Results detected 11 virus isolates by cytopathic effect, and subsequently confirmed by RT-PCR and indirect immunofluorescence. Of these, six were sequenced and identified as the lineages B.1, B.1.1, B.1.1.1, and B.1.205 according to the Pango lineage nomenclature. The prototype strain corresponded to lineage B.1.1. The analysis of the strains from the successive passages showed mutations mainly at in the spike (S) protein of the virus without variation in the identity of the lineage. Conclusion(s): Four lineages were isolated in the Vero ATCC CCL-81 cell line. Subcultures in the same cell line showed mutations in the spike protein indicating greater adaptability to the host cell and variation in pathogenicity in vitro, a behavior that allows it to have more survival success.Copyright © 2023 Anales de la Facultad de Medicina. All rights reserved.

Enhancing the sensitivity of rapid antigen detection test (RADT) of different SARS-CoV-2 variants and lineages using fluorescence-labeled antibodies and a fluorescent meter.

RT-qPCR is considered the gold standard for diagnosis of COVID-19; however, it is laborious, time-consuming, and expensive. RADTs have evolved recently as relatively inexpensive methods to address these shortcomings, but their performance for detecting different SARS-COV-2 variants remains limited. RADT test performance could be enhanced using different antibody labeling and signal detection techniques. Here, we aimed to evaluate the performance of two antigen RADTs for detecting different SARS-CoV-2 variants: (i) the conventional colorimetric RADT (Ab-conjugated with gold beads); and (ii) the new Finecare™ RADT (Ab-coated fluorescent beads). Finecare™ is a meter used for the detection of a fluorescent signal. 187 frozen nasopharyngeal swabs collected in Universal transport (UTM) that are RT-qPCR positive for different SARS-CoV-2 variants were selected, including Alpha (n = 60), Delta (n = 59), and Omicron variants (n = 108). Sixty flu and 60 RSV-positive samples were included as negative controls (total sample number = 347). The conventional RADT showed sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of 62.4% (95%CI: 54-70), 100% (95%CI: 97-100), 100% (95%CI: 100-100), and 58% (95%CI: 49-67), respectively. These measurements were enhanced using the Finecare™ RADT: sensitivity, specificity, PPV, and NPV were 92.6% (95%CI: 89.08-92.3), 96% (95%CI: 96-99.61), 98% (95%CI: 89-92.3), and 85% (95%CI: 96-99.6) respectively. The sensitivity of both RADTs could be greatly underestimated because nasopharyngeal swab samples collected UTM and stored at -80 °C were used. Despite that, our results indicate that the Finecare™ RADT is appropriate for clinical laboratory and community-based surveillance due to its high sensitivity and specificity.

Isolation and Characterization of Porcine Sapelovirus from the PDCoV-Positive Sample and Its Molecular Epidemiology in Henan Province, China

Porcine sapelovirus (PSV) is an emerging swine enteric virus that can cause various disorders including acute diarrhea, respiratory distress, reproductive failure, and polioencephalomyelitis in pigs. In this study, we isolated a PSV strain HNHB-01 from a clinical porcine deltacoronavirus- (PDCoV-) positive intestinal content of a diarrheic piglet. PSV was first identified using the small RNA deep sequencing and assembly, and further identified by the electron microscopic observation and the immunofluorescence assay. Subsequently, this virus was serially passaged in swine testis (ST) cells, and the complete genomics of PSV HNHB-01 passage 5 (P5), P30, P60, and P100 were sequenced and analyzed. 9 nucleotide mutations and 7 amino acid changes occurred in the PSV HNHB-01 P100 strain when compared with the PSV HNHB-01 P5. Pathogenicity investigation showed that orally inoculation of PSV HNHB-01 P30 could cause obvious clinical symptoms and had broad tissue tropism in 5-day-old piglets. Epidemiological investigation revealed that PSV infections and the coinfections of diarrhea coronaviruses were highly prevalent in swine herds. The complete genomes of 8 representative PSV epidemic strains were sequenced and analyzed. Phylogenetic analysis revealed that the PSV epidemic strains were closely related to other PSV reference strains that located in the Chinese clade. Furthermore, recombination analysis revealed that the recombination events were occurred in downstream of the 2C region in our sequenced PSV HNNY-02/CHN/2018 strain. Our results provided theoretical basis for future research studies of the pathogenic mechanism, evolutionary characteristics, and the development of vaccines against PSV.

Preparation and identification of broad-spectrum monoclonal antibodies against N protein of avian infectious bronchitis virus

[Objective] To prepare broad-spectrum monoclonal antibody against N protein of avian infectious bronchitis virus (IBV), so as to lay a foundation for identifying conservative domain epitope of N protein and establish a universal IBV detection method. [Method] N protein of GX-YL5, a representative strain of IBV dominant serotype in Guangxi, was expressed in prokaryote. BALB/c mice were immunized with the purified protein. After the serum titer of the immunized mice reached 104 or more, the splenocytes were fused with SP2/0 myeloma cells. After screening by indirect ELISA, monoclonal antibody was prepared by ascites-induced method. Western blotting, IFA and indirect ELISA were used to identify the titer, subtype, reaction specificity and cross-reaction spectrum. And the prepared monoclonal antibody was used for immunohistochemical detection. And the prepared monoclonal antibody was used to detect the IBV in the trachea and kidney tissues of SPF chickens artificially infected with 4 representative IBV variants (GX-N130048, GX-N160421, GX-QZ171023 and GX-QZ170728). [Result] The prepared monoclonal antibody N2D5 had a titer greater than 217 and its subtype was IgG2b. The Western blotting and IFA results showed that the monoclonal antibody N2D5 only reacted with IBV, and were negative with Newcastle disease virus (NDV), infectious laryngotracheitis virus (ILTV), avian metapneumovirus (aMPV), infectious bursal disease virus (IBDV), avian leukosis virus (ALV) and Marek's disease virus (MDV). Monoclonal antibody N2D5 reacted with many genotypes in China and all 7 serotypes of IBV currently prevalent in Guangxi, including commonly used standard strains, vaccine strains and field strains. Immunohistochemistry showed that the virus signals could be detected in the trachea and kidney tissues of SPF chickens at different time after artificial infection of 3 representative IBV strains from chicken and 1 isolated strain from duck, which further proved its broad spectrum. [Conclusion] The monoclonal antibody N2D5 of IBV prepared based on hybridoma technology belongs to the IgG2b subtype. It has the characteristics of high specificity, wide response spectrum and strong binding ability with IBV. It can be used as a specific diagnostic antibody for clinical diagnosis of IBV and the study of virus distribution.

TARGETING THE HOST-VIRUS INTERFACE TO BLOCK SARS-CoV-2 ASSEMBLY IN AIRWAY CELLS

Background: The rapid emergence of the SARS-CoV-2 Omicron variant that evades many therapies illustrates the need for antiviral treatments with high genetic barriers to resistance. The small molecule PAV-104, identified through a moderate-throughput screen involving cell-free protein synthesis, was recently shown to target a subset of host protein assembly machinery in a manner specific to viral assembly with minimal host toxicity. The chemotype shows broad activity against respiratory viral pathogens, including Orthomyxoviridae, Paramyxoviridae, Adenoviridae, Herpesviridae, and Picornaviridae, with low susceptibility to evolutionary escape. Here, we investigated the capacity of PAV-104 to inhibit SARS-CoV-2 replication in human airway epithelial cells (AECs). Method(s): Dose-dependent cytotoxicity of PAV-104 in Calu-3 cells was determined by MTT assay. Calu-3 cells were infected with SARS-CoV-2 isolate USA-WA1/2020 (MOI=0.01). Primary AECs were isolated from healthy donor lung transplant tissue, cultured at air liquid interface (ALI), and infected with SARS-CoV-2 Gamma, Delta, and Omicron variants (MOI=0.1). SARS-CoV-2 replication was assessed by RT-PCR quantitation of the N gene, immunofluorescence assay (IFA) of nucleocapsid (N) protein, and titration of supernatant (TCID50). Transient co-expression of four SARS-CoV-2 structural proteins (N, M, S, E) to produce virus-like particles (VLPs) was used to study the effect of PAV-104 on viral assembly. Drug resin affinity chromatography was performed to study the interaction between PAV-104 and N. Glycerol gradient sedimentation was used to assess N oligomerization. Total RNA-seq and the REACTOME database were used to evaluate PAV-104 effects on the host transcriptome. Result(s): PAV-104 reached 50% cytotoxicity in Calu-3 cells at 3732 nM (Fig.1A). 50 nM PAV-104 inhibited >99% of SARS-CoV-2 infection in Calu-3 cells (p< 0.01) and in primary AECs (p< 0.01) (Fig.1B-E). PAV-104 specifically inhibited SARS-CoV-2 post entry, and suppressed production of SARS-CoV-2 VLPs without affecting viral protein synthesis. PAV-104 interacted with SARS-CoV-2 N and interfered with N oligomerization. Transcriptome analysis revealed that PAV-104 treatment reversed SARS-CoV-2 induction of the interferon and maturation of nucleoprotein signaling pathways. Conclusion(s): PAV-104 is a pan-respiratory virus small molecule inhibitor with promising activity against SARS-CoV-2 in human airway epithelial cells that should be explored in animal models and clinical studies.

Jahrestagung der Gesellschaft fur Padiatrische Pneumologie.

The proceedings contain 54 papers. The topics discussed include: cytokines in severe childhood asthma;transcriptional gene regulation of interleukin-6 in epithelial cells in viral-induced asthma exacerbation;assessment of the long-term safety and efficacy of dupilumab in children with asthma: LIBERTY ASTHMA EXCURSION;impulse oscillometry bronchodilator response in preschool children;pulmonary function in non-hospitalized adults and children after mild Covid-19;exhaled aerosols in PCR-confirmed SARS-CoV-2-infected children;early respiratory infectious diseases have an influence on the gut microbiome;comparison of three eradication treatment protocols for pseudomonas aeruginosa in children and adolescents with cystic fibrosis;neutrophilic airway inflammation in children with repaired esophageal atresia-tracheoesophageal fistula (EA/TEF);and multiplex immunofluorescence and multispectral imaging as a tool to evaluate host directed therapy.

Using ex vivo bioengineered lungs to model pathologies and screening therapeutics

Introduction: More than 8 million lives are claimed annually by various respiratory diseases including lung cancer. While therapeutics is the first line of defence, treatment failure always remains challenging and research studies face a lag of transition from preclinical to clinical phase. This is partly due to the inadequate representation of the preclinical models in clinical trials. In this proof-of-concept study, we sought to use an ex-vivo model to identify lung pathologies and therapeutically screen them in a rodent model. Method(s): Briefly, the heart-lung tissues were extracted and decellularized using a detergent-based decellularization technique. Subsequently, lungs were seeded and cultured (6-10 days) with human cell lines: BEAS-2B, A549, and Calu3, demonstrating healthy lung, cancerous state, and congenital pathologies (cystic fibrosis), respectively. By altering the cultural conditions and exploiting the unique characteristics of these cell lines, we were able to model a variety of novel pathological models in ex vivo, such as advanced-stage solid tumours and the primary phase of infection via SARS-COV2. We also validated the above-mentioned observations by histology and immunofluorescence staining. Another novel part of our study includes a qualitative screening of efficacy and impact of important Therapeutics (anti-neoplastic)- Cisplatin and Wogonin, in our cancer models. Result(s): Using A549 and BEAS-2B cells, we were able to model different stages of Non-small cell lung cancer, qualitatively validated the resemblance to clinical samples and monitor the impact of different therapeutics on these models. The qualitative assessment also demonstrated different levels of cell death depending on the efficacy of the drugs. Contribution to research : Collectively this study demonstrates the remarkable versatility and strength of the ex vivo model in representing important lung pathologies and screening therapeutics in the preclinical phase.

Lassa Fever. Part 2. Laboratory Diagnostics, Treatment, Development of Medications

Globalization and high-speed means of transportation contribute to the spread of infections dangerous to humans. Airborne pathogens have pandemic potential as currently shown in case of the novel coronavirus SARS-CoV-2. Natural focal Lassa fever (LF) common in West African countries, in 35 cases was registered in non-endemic geographical areas because any person infected with Lassa virus (LASV) is a long-term source of infection (up to two months). Cases of person-to-person infection in endemic territories are described. In Germany, the facts of secondary virus transmission from patients to doctors have been recorded during the examination and blood collection from an apparently healthy person as well as during the autopsy of a deceased subjects due to severe LF course. Nonspecific malaise symptoms in LF are also characteristic of numerous other diseases common on the African continent, e.g., malaria and typhoid fever or viral infections such as yellow fever, Chikungunya, dengue and Zika, monkey pox and Ebola virus disease. In this regard, there may be similar dermatological manifestations. Timely detection of cases and differential diagnosis are crucial to ensure safe patient care and use of affordable antiviral therapy for LL provided by the drug Ribavirin. Research methods for studying LASV use polymerase chain reaction (PCR) for detecting viral RNA, electron microscopy, isolation of infectious virus cultured sensitive cells, indirect immunofluorescence reaction, enzyme immunoassay (ELISA) and immuno-chromatographic assays for the detection of antibodies and/or antigen as well as immunoblotting. Currently, test kits based on molecular and genetic methods are mainly used for LF laboratory diagnostics. Since the 1980s, ribavirin has been used to treat patients with LF. The serum accumulation of the drug in large quantities causes hemolysis, development of anemia and impaired renal function. In this regard, treatment options are being considered with decline in its concentration due to combined use with other antiviral drugs. A search for new therapeutic agents capable of inhibiting viral replication at disease early stage has been in progress due to lack of any approved vaccines.

Could SARS-CoV-2 Trigger the Formation of Antinuclear Antibodies?

Objective: The effect of severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) infection on autoimmunity in both disease and post-disease stages has not been fully explained. There is not enough information about the evaluation of autoimmune antibodies in convalescent SARS-CoV-2 patients. This study aimed to investigate the presence and types of autoantibodies in post-illness coronavirus disease-2019 (COVID-19) patients and to compare them with indirect immunofluorescence assay (IIF)-antinuclear antibody (ANA) results before SARS-CoV-2 infection. Material(s) and Method(s): Twenty-four COVID-19 patients with known and reported ANA test results prior to SARS-CoV-2 infection were included in this study. Patients' IIF-ANA, extractable nuclear antigen blot and anti-dsDNA tests were studied three and nine months after SARS-CoV-2 infection. Result(s): Three months after SARS-CoV-2 infection, 41.66% of patients had a positive IIF-ANA test. When we compared these results with pre-infection ANA results, 3 patients (12.5%) were variable. The first case was chromosomal granular positive before infection and was found to be homogeneous, and cytoplasm was speckled positive after infection. Additionally, Scl-70, DFS70, and anti-dsDNA were found to be positive. We think that lupus symptoms were triggered after COVID-19. The second case had negative ANA before infection, while the ANA was antinuclear membrane positive (2+) three months after infection. Also, anti-RNP/Sm was detected as positive. The third case had negative ANA before infection, and was detected to have speckled weakly positive ANA three months after infection. However, autoantibody positivity was not detected. Conclusion(s): As a result, these data support the idea that SARS-CoV-2 infection may trigger autoimmunity and be associated with the development of autoantibodies.Copyright © 2022 by the Turkish Society of Immunology. Turkish Journal of Immunology published by Galenos Publishing House.

'Breaking' news: A pediatric case of leukocytoclastic vasculitis after SARS-CoV-2 vaccination

Case report In May 2021, the European Medicines Agency (EMA) approved the administration of the mRNA BNT162b2 vaccine (Pfizer/BioNTech) in adolescents aged 12-15 years, in the form of a two-doses-primary course three weeks apart, with a booster dose after five months. Few adverse events have been observed in vaccinated children -mainly fever, myalgia, or local edema at the injection site. Conversely, delayed cutaneous reactions are little reported in adults and even rarer in pediatric age. A 12-year-old boy, with no previous cutaneous or autoimmune diseases nor allergic reactions to previous vaccines or drugs, came to our attention because of a cutaneous rash, which started on his right arm two days after his first dose of Pfizer/BioNTech vaccine. The rash was flat, erythematous, and purpuric, with subsequent bruise appearance and spontaneous resolution. Lesions took over on the front side of his arms and shoulders until a month after the vaccine. Urine and blood tests -including blood cell count, flow cytometry, plasma biochemistry, inflammatory index, autoantibodies, coagulation, and platelets aggregation test -did not show significant alterations. Grass pollen monosensitization was detected. Biopsy of the lesions showed 'modest perivascular lymphohistiocytic infiltrate and focal infiltration of vascular walls with swollen endothelium' with 'occasional eosinophils, scattered neutrophil granulocytes, and erythrocytes in interstitial areas,' compatible with leukocytoclastic vasculitis. Epidermidis was undamaged, and the direct immunofluorescence showed fibrinogen deposits in superficial-to-middle dermis vessels. Further tests highlighted a high immune response to the vaccine without previous Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection. No therapies were needed, and the boy experienced no other side effects undergoing his second dose. Few leukocytoclastic vasculitis cases after SARS-CoV-2-vaccination have been reported in adults, primarily flares of previously known vasculitis. A role for induced spike glycoprotein as a pseudovirion docking to specific vascular receptors has been suggested, too. To our knowledge, no similar cases were described currently, neither in such young patients nor with such atypical, focal lesions. Further studies are needed to identify the pathogenesis and possibly prevent the onset of this adverse reaction.

A Combined Method Based on the FIPV N Monoclonal Antibody Immunofluorescence Assay and RT-nPCR Method for the Rapid Diagnosis of FIP-Suspected Ascites

Feline infectious peritonitis (FIP), which is caused by feline infectious peritonitis virus (FIPV), is a fatal and immunologically mediated infectious disease among cats. At present, due to the atypical clinical symptoms and clinicopathological changes, the clinical diagnosis of FIP is still difficult. The gold standard method for the differential diagnosis of FIP is immunohistochemistry (IHC) which is time-consuming and requires specialized personnel and equipment. Therefore, a rapid and accurate clinical diagnostic method for FIPV infection is still urgently needed. In this study, based on the etiological investigation of FIPV in parts of southern China, we attempted to explore a new rapid and highly sensitive method for clinical diagnosis. The results of the etiological investigation showed that the N gene of the FIPV BS8 strain had the highest homology with other strains. Based on this, a specific FIPV BS8 N protein monoclonal antibody was successfully prepared by expression of the recombinant proteins, immunization of mice, fusion and selection of hybridoma cell lines, and screening and purification of monoclonal antibodies. Furthermore, we carried out a time-saving combination method including indirect immunofluorescence assay (IFA) and nested reverse transcription polymerase chain reaction (RT-nPCR) to examine FIP-suspected clinical samples. These results were 100% consistent with IHC. The results revealed that the combined method could be a rapid and accurate application in the diagnosis of suspected FIPV infection within 24 hours. In conclusion, the combination of IFA and RT-nPCR was shown to be a fast and reliable method for clinical FIPV diagnosis. This study will provide insight into the exploitation of FIPV N antibodies for the clinical diagnosis of FIP-suspected ascites samples.

Analysis of Covid-19 Neutralizing Antibody Levels Post Vaccination using IchromaTMand iFlash Covid-19 Nab

Background: One of the efforts to control SARS-CoV-2 infection in health workers is vaccination. In this study, the levels of SARS-CoV-2 neutralizing antibody (nAb) in health workers were measured with Ichroma and iFlash. Method(s): This study applied an observational analytic design with a prospective cohort and was conducted at Dr. Soetomo Regional Public Hospital, Surabaya, from January to November 2021. The population of this study included a total of 75 health workers after taking the second dose of the SARS-CoV-2 (Sinovac) vaccine. The Covid-19 NAb levels of the population were tested with Ichroma and iFlash on day 0 before vaccination, as well as days 14 and 28, and months 3 and 6 after vaccination. Result(s): The Friedman test indicated a significant difference in NAb levels according to the iFlash test on day 14, day 28, month 3, and month 6 compared to those before vaccination (p < 0.05). The Wilcoxon test revealed a significant difference in NAb levels on day 14, day 28, month 3, and month 6. The results of the Cochran test showed a significant difference in the positivity of NAb according to the Ichroma test on day 14, day 28, month 3, and month 6 compared to those before vaccination (p < 0.05). McNemar's test demonstrated that the COI at month 3 was not significantly different from that before vaccination;The COI at month 6 was not significantly different from those at days 14 and 28. The results of the Pearson correlation test and Bland-Altman plot indicated a moderate correlation between Ichroma and iFlash (r = 0.592, p = 0.002). Conclusion(s): Neutralizing antibodies for Covid-19 were formed after day 14 and started to increase on day 28 and started to decrease in months 3 and 6. The levels of NAb for Covid-19 were measured with Ichroma and iFlash in roughly the same pattern and had a moderate positive correlation.Copyright © 2023 Phcogj.Com.

Comparison of a rapid fluorescence immunochromatographic test with an enzyme-linked immunosorbent assay for measurement of SARS-CoV-2 spike protein antibody neutralizing activity.

BACKGROUND: SARS-CoV-2 Spike protein Receptor Binding Domain neutralizing antibodies (NAbs-RBD) inhibit the viral binding to angiotensin-converting enzyme 2 (ACE2) receptors. We compared an ELISA and a fluorescence immunochromatography (FIC) method in NAbs-RBD detection after COVID-19 immunization. METHOD: Serum samples from healthcare workers (HCWs) vaccinated with BNT162b2 were collected one and four months after the second dose. NAbs-RBD (%) detection was performed using ELISA cPass™ (FDA approved) and FIC n-AbCOVID-19® assays. RESULTS: Samples from 200 HCWs [median age (IQR): 45(35-53)] were tested with both assays. There was a good qualitative agreement between the two methods [AUC: 0.92(95%C.I.: 0.89-0.94, P-value:0.007)]. NAbs-RBD (%), one and four months after immunization, were significantly lower with FIC compared to ELISA for all age groups (P-value<0.0001). The quantitative comparison between FIC and ELISA detected slight agreement one month after the second dose [(Lin's Concordance Correlation Coefficient (CCC): 0.21(95%CI: 0.15-0.27)] which improved four months after the second dose [CCC: 0.6(95%CI: 0.54-0.66)]. CONCLUSION: FIC had good qualitative agreement with ELISA in the detection of positive NAbs-RBD (%) and could be an alternative for rapid NAbs-RBD (%) testing.

Severe epidermolysis bullosa acquisita in a patient with graft-versus-host disease: causal or coincidental?

Graft-versus-host disease (GvHD) is common after haematopoietic cell transplantation (HCT). Mucocutaneous manifestations are variable and may simulate autoimmune bullous dermatoses. However, the association of GvHD with autoimmune disorders, including bullous dermatoses, is also well recognized. We describe a patient with GvHD in whom severe and relapsing epidermolysis bullosa acquisita (EBA) was diagnosed 3 years after transplant and propose a causal association with GvHD. A 66-year-old woman developed GvHD following allogeneic HCT for acute myeloid leukaemia in 2016. This affected her gastrointestinal tract and skin but improved with oral corticosteroids and ciclosporin. In 2019 she presented with a widespread rash consisting of large, tense, haemorrhagic blisters. Histological features were in keeping with EBA. Direct immunofluorescence was also consistent with EBA, demonstrating linear positivity for IgG and C3 confined to the blister base, as was detection of collagen VII antibodies on indirect immunofluorescence. She was admitted and treated with high-dose oral steroids, ciclosporin and intravenous immunoglobulin (IVIg) with eventual resolution of blistering. Although further IVIg administration was planned as an outpatient, this coincided with the start of the COVID-19 pandemic and she elected not to attend and also stopped all medication. Despite this, her EBA remained quiescent until September 2021 when she was readmitted with a severe deterioration in blistering and significant dysphagia due to an oesophageal stricture, with a weight of 31.7 kg. Once again, she responded rapidly to oral prednisolone and IVIg. Dapsone was considered but precluded by G6PD deficiency and there were clinical and adherence concerns about using mycophenolate mofetil. Upon discharge she was again nonadherent to medication and failed to attend for planned IVIg. She flared and was admitted for a third time in December 2021, requiring gastrostomy for nutritional support;her weight at this time was 26.4 kg. Her EBA is currently well controlled on prednisolone and IVIg. EBA is a rare, acquired blistering disorder secondary to autoantibodies targeting type VII collagen. Previous studies have found circulating basement membrane zone (BMZ) antibodies in 24% of chronic GvHD patients, possibly generated in response to chronic BMZ damage (Hofmann SC, Kopp G, Gall C et al. Basement membrane antibodies in sera of haematopoietic cell recipients are associated with graft-versushost disease. J Eur Acad Dermatol Venereol 2010;24: 587-94). Corresponding clinical manifestations are rare, with bullous pemphigoid the most frequently reported. EBA is much less common with four previously reported cases [Brassat S, Fleury J, Camus M, et al. (Epidermolysa bullosa acquisita and graftversus- host disease). Ann Dermatol Venereol 2014;141: 369-73 (in French)]. As a fifth case of EBA, our patient provides further evidence of a likely pathophysiological relationship between GvHD and autoimmune subepidermal bullous dermatoses, and highlights the significant challenges of managing these vulnerable patient groups during the COVID-19 pandemic.

A Case of Iga Vasculitis That Developed Gross Hematuria and Lower-Limb Purpura Following Covid-19 Vaccines

Introduction: There have been some reports on flare-ups of kidney diseases following COVID-19 vaccines such as IgA nephropathy and minimal change disease. However, there have been few reports on those of IgA vasculitis following the vaccines yet. We report a case of IgA vasculitis with a flare-up of gross hematuria and lower-limb purpura following Moderna COVID-19 vaccines. Method(s): The patient is a 16-year-old female with no previous history of abnormal results of urinalyses before April in 2021. She had developed microscopic hematuria, proteinuria and purpura on both of her lower limbs that emerged and then disappeared repeatedly since then. She received Moderna COVID-19 vaccines in August and September in 2021, both of which were followed by gross hematuria lasting for around 10 days. The lower-limb purpura reemerged at the same time as the hematuria. Microscopic hematuria of around 30-49 RBC/HPF, glomerular hematuria of moderate degree and urine protein-to-creatinine ratio (UPCR) of around 0.8 g/gCr had continuously been detected. Skin and kidney biopsies were performed in December in 2021 and in February in 2022 respectively. Result(s): The skin tissue showed formation of leukocytoclastic vasculitis, and the kidney tissue showed that of cellular and fibrocellular crescents and endocapillary hypercellularity. Immunofluorescence staining of both tissues showed deposition of galactose-deficient IgA1(Gd-IgA1) and C3, and she was diagnosed as IgA vasculitis. She received steroid pulse therapy followed by tonsillectomy. The lower-limb purpura has disappeared after she received three courses of the steroid pulse therapy, but microscopic hematuria and UPCR of around 0.8 g/gCr have still continued. Conclusion(s): IgA vasculitis is leukocytoclastic vasculitis characterized by deposition of Gd-IgA1 on microvessel walls in skin and on glomerular capillaries in kidneys. The detailed mechanism of IgA vasculitis has not been fully elucidated yet. Gross hematuria following an upper respiratory infection is considered as a characteristic clinical symptom of IgA vasculitis as well as IgA nephropathy. Post-vaccination gross hematuria of patients with IgA nephropathy has been reported, and it is believed that innate immunity is related to its mechanism. Moderna COVID-19 vaccines, which the patient received, are mRNA vaccines. We estimate that exposure to the mRNA vaccine triggered excess glomerular deposition of Gd-IgA1-containing immune complexes and subsequent gross hematuria by overactivation of innate immunity such as Toll-like receptors that detect RNA. This case suggests that such immune activation by a mRNA vaccine might be related not only to the mechanism of IgA nephropathy but also to that of IgA vasculitis. No conflict of interestCopyright © 2023

Perindopril Reduce SARS-COV-2 Spike-ACE2 Binding, ACE2 Overexpression and Cytokine Storm in Adipocyte Cell Infected with SARS-COV-2

Background and Aims: The SARS-CoV-2 virus can infect adipocyte cells via ACE2 Receptor thus triggering ACE2 overexpression and cytokine storms which cause lethal complications. Hence, we explore the effect of Perindopril on the expression of ACE2, IL-6, IL-1B TNF-alpha in adipocyte cultures infected by SARS-CoV-2 spike protein and identify the possible mechanism involved. Material(s) and Method(s): Adipocyte culture obtained from a healthy and obese donor was divided into 4 triplicate groups (P0: negative control without treatment;P1: positive control (SARS-CoV-2 spike protein);P2: SARS-CoV-2 spike protein + exposure to Perindopril 0.5 muM);P3: SARS-CoV-2 spike protein + anti-ACE2 antibody 100 mug/mL;and evaluated at 24 and 48 hours. ACE2 expression was evaluated using immunofluorescence. IL-6, TNFalpha, and IL-1B were evaluated using ELISA. SARS-COV-2 Spike-ACE2 Binding was evaluated using Competitive ELISA. Data analysis was performed using SPSS 25.0 software. Result(s): At first 24 hours of incubation, perindopril treatment has the highest ACE2 expression compared to negative control, positive control and anti-ACE2 antibody (113.52+/-0.34 ng/mL vs 13.3+/-0.87 ng/mL, 90.2+/-2.73 ng/mL, 17.3+/-0.11 ng/mL, p<0.01), lower ACE-ACE2R binding compared to anti-ACE2 antibody group (169.52+/-4.07 ng/mL vs 290.71+/-6.22 ng/mL, p<0.01) and higher IL-6 expression compared to positive control group (64.65+/-0.12 ng/mL vs 60.08+/-0.77 ng/mL p<0.01). Interestingly, after 48 hours, perindopril treatment was shown to prevent further increase of ACE2 expression compared to a positive control (47.37+/-0.76 ng/mL vs 80.31+/-5.37 ng/mL, p<0.01), higher SARS-COV-2 Spike-ACE2 binding compared to anti-ACE2 antibody group (143.68+/-3.68 ng/mL vs 103.1+/-9.49 ng/mL, p<0.01), and lower IL-6 expression compared to the positive control group (42.66+/-1.94 ng/mL vs 90.93+/-2.48 ng/mL p<0.01). However, no significant difference in TNFalpha and IL-1B expression between perindopril treatment and positive control in both 24 and 48 hours. Conclusion(s): This study showed that perindopril reduces cytokine storm by preventing ACE-2 and IL-6 overexpression via an increasing number of SARS-COV-2 Spike-ACE2 competitive binding in adipocyte culture infected with SARS-COV-2 spike protein. A further clinical trial is needed to prove the benefit of perindopril in obese patients with COVID-19.

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.

COVID-19: From the Molecular Mechanisms to Treatment

The 2019 novel coronavirus (SARS-CoV-2) causes severe pneumonia called COVID-19 and leads to severe acute respiratory syndrome with a high mortality rate. The SARS-CoV-2 virus in the human body leads to jumpstarting immune reactions and multi-organ inflammation, which has poorer outcomes in the presence of predisposing conditions, including hypertension, dyslipidemia, dysglycemia, abnormal adiposity, and even endothelial dysfunction via biomolecular mechanisms. In addition, leucopenia, hypoxemia, and high levels of both cytokines and chemokines in the acute phase of this disease, as well as some abnormalities in chest CT images, were reported in most patients. The spike protein in SARS-CoV-2, the primary cell surface protein, helps the virus anchor and enter the human host cells. Additionally, new mutations have mainly happened for spike protein, which has promoted the infection's transmissibility and severity, which may influence manufactured vaccines' efficacy. The exact mechanisms of the pathogenesis, besides molecular aspects of COVID-19 related to the disease stages, are not well known. The altered molecular functions in the case of immune responses, including T CD4+, CD8+, and NK cells, besides the overactivity in other components and outstanding factors in cytokines like interleukin-2, were involved in severe cases of SARS-CoV-2. Accordingly, it is highly needed to identify the SARS-CoV-2 bio-molecular characteristics to help identify the pathogenesis of COVID-19. This study aimed to investigate the bio-molecular aspects of SARS-CoV-2 infection, focusing on novel SARS-CoV-2 variants and their effects on vaccine efficacy.Copyright © 2022 NRITLD, National Research Institute of Tuberculosis and Lung Disease, Iran.

Phototherapy to treat a COVID-19 vaccine reaction

A 59-year-old man presented with a widespread morbilliform rash after receiving the second dose of the Pfizer-BioNTech COVID-19 mRNA vaccine. He had no significant medical history and no known allergies. He did not take any regular medication. He developed pruritus without rash 4 h after his first vaccine. This resolved after 10 days without intervention. One day after his second dose, he developed an extensive pruritic morbilliform eruption on his trunk and limbs, affecting 35% of his body surface area. with no mucous membrane involvement. The rash persisted for 4 weeks after his second vaccination and he was referred to dermatology. Eosinophils were raised at 0.54 and liver function tests were normal. Antinuclear antibodies and extractable nuclear antigen were negative. Complement levels were normal. Histology showed mild epidermal acanthosis, spongiosis and subcorneal vesicles. Within the superficial to mid-dermis, there was a mixed chronic inflammatory infiltrate comprising lymphocytes, plasma cells, neutrophils and numerous eosinophils. Direct immunofluorescence was negative. He received a tapering dose of oral prednisolone with mometasone topically. Despite substantial improvement with this regimen, his rash began to worsen 2 days following discontinuation of oral prednisolone. He was still using daily mometasone on cessation of oral steroids. He was trialled on oral doxycycline for 1 month, which led to a marked improvement in the morbilliform rash. Despite improvement in the rash, the patient reported ongoing intense daily pruritus which was having a marked impact on his quality of life. He has commenced on narrowband ultraviolet B (UVB) phototherapy to treat his persistent pruritis, with good effect to date. Morbilliform eruptions have been reported as a cutaneous manifestation of COVID-19 and as a side-effect of mRNA vaccines. Proposed mechanisms for the development of skin rashes post-mRNA vaccines include viral protein expression following vaccination, prior infection with COVID-19 causing cross-reaction with the mRNA vaccine encoded antigen and vaccine components acting as haptens inducing a T helper 2 inflammatory reaction characterized by interleukin (IL)-4 and IL-13 expression. Drug-induced maculopapular eruptions typically resolve within 7-14 days on withdrawal of the culprit medication. The persistent nature in our patient may imply a complex immune response. The use of phototherapy to treat inflammatory dermatoses and pruritic conditions such as nodular prurigo is well described. The antipruritic effect of phototherapy is thought to work via modulation of both the neural pathways involved in itch and local immune cells in the skin. Our case highlights that phototherapy can be used in the treatment of cutaneous side-effects that arise after COVID-19 vaccines. To the best of our knowledge, this case is one of the first to use narrowband UVB phototherapy to treat a cutaneous side-effect of an mRNA vaccine.