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Background: Many mechanisms responsible for COVID-19 pathogenesis are well-known, but COVID-19 includes features with unclear pathogenesis, like autonomic dysregulation, coagulopathies, and high levels of inflammation. The SARS-CoV-2 spike protein receptor binding domain (RBD) receptor is angiotensin converting enzyme 2 (ACE2). We hypothesized that some COVID-19 patients may develop immunoglobulins (Igs) that have negative molecular image of RBD sufficiently similar to ACE2 to yield ACE2-like catalytic activity - ACE2-like 'abzymes'. Method(s): To explore this hypothesis, we studied 67 patients hospitalized with COVID-19 who had disodium ethylenediaminetetraacetate (EDTA) anticoagulated plasma samples available, obtained about 7 days after admission. We used commercially available fluorometric ACE2 assays (Abcam), and a SpectraMax M5 microplate reader (Molecular Devices), measuring Relative Fluorescent Unit (RFU, Ex/Em = 320/420 nm;RFU) in a kinetic mode every 20 min at 37C. ACE2 inhibitor provided in the assay kit was used for additional controls. In some control experiments, we added Zn2+ to plasma, or conducted serial dilutions to decrease Zn2+. To deplete Igs, we passed plasma samples through a 0.45 mum filter to remove large particles, then passed the material through 100kDa cut-off ultrafiltration membrane (PierceTM) columns, and finally used protein A/G Magnetic Beads (Life Technologies) to specifically deplete Ig, removing >99.99% of Ig as assessed with a human IgG ELISA Kit (Abcam). Result(s): ACE2 is a metalloprotease that requires Zn2+ for activity. However, we found that the plasma of 11 of the 67 patients could cleave a synthetic ACE2 peptide substrate, even though the plasma samples were collected using EDTA anticoagulant. When we spiked plasma with synthetic ACE2, no ACE2 substrate cleavage activity was observed unless Zn2+ was added, or the plasma was diluted to decrease EDTA concentration. After processing samples by size exclusion and protein A/G adsorption, the plasma samples did not cleave the ACE2 substrate peptide. Conclusion(s): The data suggest that some patients with COVID-19 develop Igs with activity capable of cleaving synthetic ACE2 substrate. Since abzymes can exhibit promiscuous substrate specificities compared to the enzyme whose active site image they resemble, and since proteolytic cascades regulate physiologic processes, anti-RBD abzymes may contribute to some otherwise obscure features of COVID-19 pathogenesis. (Figure Presented).
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Increased levels of neutrophil extracellular traps (NETs) have been detected in individuals with vaccine complications after the ChAdOx1 nCov vaccine with a correlation between the severity of vaccine side effects and the level of NETosis. DNases may disrupt NETs by degrading their content of DNA, and a balance has been reported between NETs and DNases. Because of this and since the inflammatory marker NETs may be used as a confirmatory test in diagnosing VITT, it is of interest to monitor levels of DNase in patients with increased NETs levels. The current novel rapid DNase ELISA was tested in blood samples of patients with known increased levels of NETs with or without VITT after ChAdOx1 nCoV-19 vaccination. DNase levels in VITT patients were significantly increased compared with normal unvaccinated blood donors and compared with patients with post-vaccination symptoms but not VITT. However, since EDTA was found to inhibit DNase, serum and not EDTA-plasma samples should be applied for DNase testing. The novel DNase assay may serve as a supplementary test to the NETs test when analysing samples from patients with suspected increased NETs levels.Copyright © 2023 The Scandinavian Foundation for Immunology.
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Background: According to data from the World Health Organization updated to September 30, 2022, SARS-CoV-2 is a viral disease, that infected six hundred million people and claimed six and a half million victims in all over the world. Some authors describe the alteration of blood parameters in pediatric age following COVID-19 such as: anomalies of the leukocyte formula, CK-MB and LDH enzymes, C reactive protein (PCR) and procalcitonin (PCT). Based on these studies we wanted to focus on two cardiac biomarkers: type B natriuretic peptide (BNP) and high sensitivity cardiac troponin (cTnT-hs). In order to suggest a strategy aimed at preventing the possible occurrence of cardiovascular complications in children we monitored them in patients with specific anti-SARS-CoV-2 immunoglobulins (IgG). Method(s): From November 2020 to December 2021, 53 children with positive IgG to SARS-CoV-2 by immunochromatographic method were tested for BNP and cTnT-hs. Samples collected in potassium EDTA tubes were tested for BNP by a fluorescence immunoassay. cTnT-hs was determined with an electrochemiluminescence sandwich test, which employs two specific monoclonal antibodies directed against cardiac troponin. The BNP cutoff is 100 pg/mL for patients aged 1 to 14 years, while that of cTnT-hs is 14 ng/L. All patients were monitored from hospitalization to discharge. Result(s): At least one of the two biomarkers resulted pathologic in 35 (66%) patients, while 18 (34%) were negative. The 35 pathologic cases were distributed as follows: 12 were simultaneously positive for cTnT-hs and BNP;13 were only positive for BNP, and 10 only positive for cTnT-hs. Sick children had a BNP mean value of 634 pg/mL at hospitalization, and of 94 pg/mL at discharge. For cTnT-hs, the initial mean value was 46 ng/l and the final was 17 ng/L. Twelve children with both altered values were monitored for a longer time, as they returned negative more slowly. Repeated dosages of the two analytes from hospitalization to discharge, showed a constant decrease over the time. After about ten days from the first dosage, both parameters returned to values near to the relative cutoffs. Conclusion(s): Measuring BNP and cTnt-hs can be useful for screening and monitoring pediatric patients positive for anti-SARSCoV-2 IgG.Copyright © 2022 EDIZIONI MINERVA MEDICA.
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Background: Infections with SARS-CoV- 2 cause the coronavirus disease 2019 (COVID-19) pandemic. Alterations in immune cells of COVID-19 patients may predict the subsequent severity of disease. The changes in composition of immune cells in COVID-19 patients include lymphopenia, lower neutrophil to lymphocyte-ratios and an eosinopenia in about 50 to 80% of hospitalized patients. Eosinophils and neutrophils can interact with T cells via immune checkpoints receptors such as programmed death (PD)-1 on T cells and its counterpart PD-ligand 1 (PD-L1) on eosinophils or neutrophils. There are only limited studies on PD-1 and PD-L1 expressions in viral infections, we aimed to elucidate the interplay of T cells and other peripheral cells by analysing the immune checkpoints PD-1 and PD-L1 in expression during COVID-19. Method(s): Using flow cytometry, we have now analysed the immune checkpoint receptor expressions on whole blood cells from a total of 38 COVID-19 patients. The patient cohort comprises all ages and both sexes with the disease severity ranging from mild, moderate to severe, with ~10% mortality. We have further been investigating 21 biomarkers (G-CSF, GM-CSF, IFN-gamma, TGF-beta1, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12p70, IL-13, IL-17A, IL-18, IL-23, IL-33, IP-10, MCP-1, MIP-1beta, TNF-alpha, and YKL-40) in plasma on a cohort of 76 COVID-19 patients using the MesoScale Multiplex Assay platform, with 48 healthy controls. Result(s): PD-L1 expression on eosinophils was significantly lower in COVID-19 patients in initial stages of infection, relative to healthy controls. There was an inverse relationship between disease progression and the expression of PD-1 on CD8+ T cells. These data suggests that analysis of PD-L1- PD1 cell networks in immune cells of EDTA blood of COVID-19 patients can predict disease outcomes. While most detectable biomarkers are strongly increased in COVID samples overall compared to healthy controls, the more severe the disease the higher the blood biomarker concentration. Conclusion(s): Taken together, the analysis of PD-L1- PD1 cell networks in immune cells together with plasma biomarkers of COVID-19 patients can predict disease outcomes.
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Introduction/ Background: Because of limitations of the RT-PCR for the diagnosis of SARS-CoV-2 for large-scale testing, data based on such a molecular method may not reflect the actual exposure to the virus. In this study, we assessed the seroprevalence of SARS-CoV-2 during the first wave of the SARS-CoV-2 pandemic in Senegal. Methods: A total of 3,978 samples were collected from nine regions of Senegal between July and September 2020. Participants were recruited from the Dakar, Thiès, Diourbel, Louga, Saint Louis, Kaolack, Ziguinchor, Saint-Louis, and Tambacounda which are the main regions of Senegal. From each participant, EDTA blood was collected and approximately 2ml of plasma were stored at 80°C for the subsequent anti-IgG and anti-IgM qualitative test using the lateral flow Healgen IgG/IgM SARS-CoV-2 test, which has previously been successfully evaluated. Results: Our data have shown a national SARS-CoV-2 prevalence of 28% during the first wave. The highest exposure to the virus was found in Ziguinchor with a prevalence of nearly 60% while the Thiès, Louga and Kaolack regions elicited the lowest prevalence. Considering the age groups [0-18], [19-40], [41-60] and > 60 years, there was no significant difference in the seroprevalence despite a slight predominance among [19- 40] years. In Dakar, no significant difference in prevalence between the four departments. No significant difference in exposure to SARS-CoV-2 between males and females was found despite a slight predominance in males. Impact: The seroprevalence eliciting the exposure of a given population to SARS-CoV-2 could provide important information that can serve not only for prevention guidances for control programmes but also SARSCoV- 2 vaccine strategies. Conclusion: In the light of these results, it appears that the circulation of the SARS-CoV-2 in Senegal during the first pandemic wave was much higher than what was reported at the national level based on RT-PCR testing.
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Background: The new Severe Acute Respiratory Syndrome Virus-2 (SARS-CoV-2), which is responsible coronavirus disease (COVID-19), spread worldwide from China, causing a pandemic from late December 2019. Due to the high proportion of asymptomatic or mild infections (approximately 80%), data restricted to laboratoryconfirmed cases do not capture the true extent of the spread or burden of the virus, or its infection-fatality ratio. Therefore, serological detection of specific antibodies against SARS-CoV-2 can better estimate the true number of infections. The current study aimed to estimate the seroprevalence of SARS-CoV-2 antibodies among the whole blood donors without any prior COVID-19 history or symptoms. Aims: To determine seroprevalence of SARS-CoV-2 (COVID-19) antibody (IgG and IgM) among asymptomatic healthy blood donors. Methods: This was a cross sectional study conducted between March 16 and July 10, 2021 among 300 blood donors without any prior COVID-19 history or symptoms who came to a tertiary care, multispecialty hospital in south India. Any donor who had travelled abroad or came back from abroad after January,2020 and donors who had received COVID-19 vaccine are excluded from the study. 3 ml venous blood was drawn in EDTA tube from participants and was tested by 'Access SARS CoV-2 IgG assay' and 'Access SARS CoV-2 IgM assay' by UniCel DxI 800 Immunoassay analyser (Beckman coulter). The Access SARS CoV-2 IgG assay and the Access SARS Cov-2 IgM assay detect antibodies to the Receptor Binding Domain (RBD) of the Spike Protein. Result was reported as Reactiveif Signal/Cut-off (S/CO) > 1.0 and non-Reactive if S/CO <1. Data was collected and entered into excel sheets and was analysed by using the software SPSS version 25. Results: A total of 300 healthy blood donors were included. All were males with the mean age being 26.98 years. The study reported seroprevalence of 15.3% for IgG and 4.3% for IgM (95%CI) among whole blood donors. Seroprevalence was similar across age groups, diet patterns, voluntary/ replacement donations, area of residence, ABO and Rh groups without any statistical significance. However higher IgG response was noted in the 30-45 age group and among B Positive blood group donors. Summary/Conclusions: Almost 15% of blood donors were seroconverted for COVID-19 during second wave. This is a reflection of widespread seroprevalence in the adult male population. Real-time seroprevalence studies will help to know the herd immunity among the blood donors which will assist in knowing the Covid-19 transmission dynamics, distribution of immunity levels at a particular point in time, immunity gaps, development of novel therapeutics and prioritize the vaccination programmes to high-risk individuals.
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Objetivos: Investigar, no Hemocentro Regional de Santa Maria (HEMOSM), os casos de doadores de sangue D negativos que apresentaram em sua fenotipagem resultados positivos para os antígenos (AG) C, E e Kell-1 na pesquisa de AG eritrocitários C.c,E,e e Kell-1. Material e métodos: Este é um estudo observacional retrospectivo realizado através da coleta de dados do Sistema HEMOVIDA (Sistema Nacional de Gerenciamento em Serviços de Hemoterapia) e dos arquivos do Laboratório de Imunohematologia do HEMOSM durante o período de fevereiro/2020 a julho/2021. As amostras utilizadas para o ensaio são amostras de sangue coletadas em tubo com anticoagulante EDTA, sendo centrifugadas e posteriormente tendo volume do concentrado de hemácias empregado na preparação de suspensões para realização dos testes conforme instruções dos fabricantes. A técnica empregada foi a aglutinação-centrifugação em cartão com gel. Anticorpos monoclonais dirigidos aos AG pesquisados encontram-se suspensos no gel em diferentes microtubos. Resultados: O total de doações com tipagem sanguínea D(-) foi de 2299 doações. Destas, 351 doadores (aproximadamente 15%) apresentaram um ou mais dos AG C, E ou Kell-1. Dentre estes, 139 doadores apresentaram fenótipo Rh Ccee, 29 apresentaram fenótipo ccEe, ainda 21 apresentaram ambos os AG, com fenótipo CcEe, enquanto o total de doadores com pesquisa positiva para o AG Kell-1 foi de 162. Discussão: Indivíduos que apresentam a tipagem sanguínea com resultado D(-), usualmente apresentam fenótipo Rh ccee com K(-), ou seja, sem a presença de AG C, E e Kell-1. Para doadores e para pacientes (pcte), a pesquisa destes AG em laboratórios de imunohematologia é importantíssima, isto porque C, E e Kell-1 apresentam elevada antigenicidade e, portanto, risco de sensibilização no caso de transfusão de hemácias com estes AG em pcte que não os apresentam. Sendo assim, concentrados de hemácias (CH) com D(-) que apresentem C, E e/ou Kell-1 devem ser direcionados para pcte que apresentem estes AG, na tentativa de utilização do hemocomponente, especialmente no período da pandemia de COVID-19, quando as doações de sangue reduziram aproximadamente 10% no HEMOSM durante o primeiro ano da pandemia (em comparação com o mesmo período do ano anterior). A destinação racional destes hemocomponentes no sentido de impedir a sensibilização dos pcte é essencial para que em futuras provas de compatibilização estes pcte não corram o risco de apresentar incompatibilidade decorrente da formação de anticorpos irregulares Anti-C, Anti-E e Anti-Kell-1. Conclusão: A utilização de CH D(-) com a presença de C, E e/ou Kell-1 só pode se dar no caso de os pcte apresentarem prova cruzada e configuração antigênica compatíveis com as hemácias do doador. Em razão disso, alguns desses CH podem permanecer represados nos estoques de sangue até seu vencimento e descarte. Uma alternativa para a utilização destes CH é a sua destinação para a compatibilização e uso em pcte D+, uma vez que estes apresentam AG C, E e/ou Kell-1 com maior frequência. Assim, são transfundidas hemácias com configuração antigênica compatível na tentativa de destinação desses CH para que não cheguem a ser desprezadas por validade, especialmente no contexto da redução das doações.
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Die SARS-CoV-2-Pandemie konfrontiert die Transplantationsmedizin mit besonderen Herausforderungen. Neben einem stets den infektiologischen Gegebenheiten angepasstem Transplantationsprogramm gilt es, das Risiko für Nierentransplantierte bestmöglich zu reduzieren. Insgesamt sind Nierentransplantierte aufgrund ihrer kardiovaskulären Komorbiditäten und ihrer Immunsuppression als hochrisikobehaftet anzusehen. Die ERA-EDTA bietet ein differenziertes Stufenschema zum Management der Immunsuppression während einer SARS-CoV-2-Infektion an. Die humorale und zelluläre Immunantwort auf eine Coronaschutzimpfung ist im Vergleich zu Immunkompetenten eingeschränkt. Aktuelle Untersuchungen zeigen einen deutlichen Immunisierungsbenefit durch den Einsatz einer Booster-Impfung.
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Background: Serology tests for detecting the antibodies to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can identify previous infection and help to confirm the presence of current infection. Objective: The aim of this study was to evaluate the performances of a newly developed high throughput immunoassay for anti-SARS-CoV-2 IgG antibody detection. Results: Clinical agreement studies were performed in 77 COVID-19 patient serum samples and 226 negative donor serum/plasma samples. Positive percent agreement (PPA) was 46.15% (95% CI: 19.22% ∼74.87%), 61.54% (95% CI: 31.58% ∼86.14%), and 97.53% (95% CI: 91.36% ∼99.70%) for samples collected on 0-7 days, 8-14 days, and ≥15 days from symptom onset, respectively. Negative Percent Agreement (NPA) was 98.23% (95% CI: 95.53% ∼99.52%). No cross-reactivity was observed to patient samples positive for IgG antibodies against the following pathogens: HIV, HAV, HBV, RSV, CMV, EBV, Rubella, Influenza A, and Influenza B. Hemoglobin (200 mg/dL), bilirubin (2 mg/dL) and EDTA (10 mM) showed no significant interfering effect on this assay. Conclusion: An anti-SARS-CoV-2 IgG antibody assay with high sensitivity and specificity has been developed. With the high throughput, this assay will speed up the anti-SARS-CoV-2 IgG testing.
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Background The SARS-CoV-2 pandemic has assaulted all aspects of daily life. Medical professionals in oncology face additional challenges with balancing prompt cancer diagnosis and urgent treatment against potential COVID-19 exposure risk in these high-risk patients. We designed this prospective freewill study to offer testing for SAR2-CoV-2 viral RNA and/or anti-COVID-19, respectively in asymptomatic medical and research staff who work in direct contact with cancer patients. The overall goal was to evaluate the prevalence of infection in this group of asymptomatic healthcare providers to reduce exposure of cancer patients to asymptomatic staff. Methods Asymptomatic medical and research staff who work in direct contact with cancer patients were asked to voluntarily be tested for either SARS-CoV-2 viral RNA or antibodies or both. Either NP swabs and/or blood samples (EDTA tube) were collected. Tests are performed at Sinochips Kansas LLC, Sinochips Diagnostics (CLIA number:17D2176068, CAP number: 8709463). The PCR test is performed with FDA authorized 2019-Novel Coronavirus (2019-nCoV) Real-Time RT-PCR Diagnostic Panel EUA. The Elecsys® Anti-SARS-CoV-2 (Roche Diagnostics) immunoassay was used to qualitative detection of antibodies to SARS-CoV-2 in human plasma. Results From 06/18/2020 to 12/18/2020, 861 participated in the study. 1095 tests were completed for SAR2-CoV-2 virus infection, and 918 were completed for antibody. Amount participants, 530 had both virus and antibody tested. 235 were tested more than once for viral infection and 166 were tested more than once for the antibody. Median age of participants was 39 years (IQR 32-51 years). Among these 84.7% were females, 84.4% white, 6.7% African American, 4.8% Asian and 84.7% non-Hispanic. The cumulative incidence of a positive test for the virus was 2.2% (16/712), and for the antibody test was 3.8% (26/679). 5 had both viral and antibody tests positive, with an average time of 4.1 weeks from viral testing positivity to detectable antibody among 3 cases and 2 cases with both viral infection and antibody detected at same time. There were 3 cases virus was detected more than once after turning positive. 2 remained positive at 16 and 22 days after initial test and one turned negative at 36 days as of last follow up. There were 7 cases where the antibody was tested more than once after turning positive and all 7 remained positive as of last follow up (range 7-103 days). Conclusion Prospective voluntary testing in asymptomatic medical and research staff who work in direct contact with cancer patients was feasible and resulted in identification of asymptomatic carriers who then placed in quarantine, thereby limiting exposure to cancer patients. Medical and research staff who work with cancer patients are general very cautious and the frequency of infections were significantly lower than general society. In addition, it seems that 1) virus and antibody may co-exist in the same person after exposure, and 2) the antibody may last for a relatively long time.
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BACKGROUND AND AIM: Multiparametric flow cytometry method is considered as the gold standard for the determination of lymphocyte subgroup analysis. In this study, it was aimed to perform lymphocyte subgroup analysis in patients with COVID-19 and to compare it with the healthy group. METHODS: The study included 50 patients with COVID-19 who applied to Diskapi Yildirim Beyazit Training and Research Hospital after approval of the ethics committee. All COVID-19 patients (n=50) and healthy controls (n=30) are equal in age and gender. Whole blood samples were taken into an EDTA tube and measured by a hemogram analyzer within 2 hours. Lymphocyte subgroup analyses (CD3, CD4, CD8, CD19, CD56, CD16) and activated T lymphocytes(HLADR) were performed using the flow cytometric method in the same samples. Lymphocyte counts were calculated using the dual platform. RESULTS: White blood cell and lymphocyte counts were significantly low in the patients with COVID- 19 (respectively, p=0.036). Flow cytometric analysis revealed that the CD3+ T lymphocyte counts and CD19+ B lymphocytes counts and percentage were significantly lower (p=0.008, <0.001, 0.004) in disease group compared to the controls but no difference observed in NK cells. In T lymphocytes, CD4+ T and CD8+ T lymphocyte counts were significantly lower (p=0.007, <0.05), but their percentages and CD4/CD8 ratios was not significantly different. The percentage of HLADR expression in T lymphocytes was significantly increased compared to the healthy group (p=0.001). CONCLUSIONS: T and B lymphocyte counts were low in COVID 19 patients. Activated T lymphocytes may be involved in the pathogenesis of the disease.
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Background. Tissue donors are evaluated for communicable disease in order to minimize the risk of transmission to recipients. Although there are data suggesting SARS-CoV-2 viremia across a wide spectrum of illness, prevalence in deceased tissue donors and the potential for transplant transmission are unknown. Methods. Eight tissue banks participated in a retrospective analysis of samples from eligible deceased tissue donors from Oct 2019 through June 2020, one participant in Canada and the remainder located in the United States. All four Census regions of the continental US and all major racial-ethnic groups were represented. EDTA or sodium citrate plasma aliquots were tested in singlicate with the Research Use Only Procleix SARS-CoV-2 Assay on the Procleix Panther System, which uses transcription-mediated nucleic acid amplification (TMA) technology for detection of the SARSCoV-2 RNA. Plasma (or if unavailable, serum) aliquots were sent to Grifols for an alternate SARS-CoV-2 nucleic acid amplification (NAT) test to verify reactivity and also sent for antibody testing using the emergency use authorization Ortho VITROS Immunodiagnostic Products Anti-SARS-CoV-2 Total test. The VITROS assay uses immunometric technology for qualitative measurement of total antibody (IgG, IgA and IgM) to SARS-CoV-2. The proportion of donors with confirmed RNAemia (i.e., presence of SARS-CoV-2 RNA in plasma or serum) and 95% confidence intervals were computed. Results. Of 3,455 donor samples with valid final results, 26 (0.76%) were initially positive for SARS-CoV-2 RNA;of these, 3 were confirmed by alternate NAT. Of donor samples collected in 2019 0.00% (95% CI: 0.00%,0.43%) were confirmed RNAemic, while of those collected in 2020, 0.12% (0.04%,0.34%) were confirmed RNAemic. One of 26 initial positive, and none of the three samples confirmed by alternate NAT, tested positive for anti-SARS-CoV-2 Spike antibodies by serology. Infectivity studies are pending on one sample with sufficient available volume. Conclusion. The rate of SARS-CoV-2 RNAemia in deceased tissue donors is approximately 1 per 1,000, and it is unknown whether this RNAemia reflects the presence of infectious virus. Given these results, the risk of transmission through tissue is most likely to be low.
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Background. The spread of carbapenem resistant Pseudomonas aeruginosa and carbapenemase-producing Enterobacterales (CPE) has had a great impact on morbidity and mortality. COVID-19 pandemic has favoured the selection of these microorganisms because of the excessive and prolonged use of broad-spectrum antibiotics and the outbreaks related to patient transfer between hospitals and inadequate use of personal protective equipment. Therefore, detection is considered essential for their control. Our aim was to compare conventional phenotypic synergy tests and two lateral flow immunoassays for detecting carbapenemases in Enterobacterales and P. aeruginosa. Methods. We analysed 100 carbapenem-resistant Gram-negative bacilli isolates, 80 Enterobacterales and 20 Pseudomonas aeruginosa, (86 isolates producing KPC, NDM, OXA-48, IMP and VIM carbapenemases and 14 non-carbapenemase-producing isolates). We performed a modified Hodge test, boronic acid and ethylenediaminetetraacetic acid (EDTA) synergy tests, and two lateral flow immunoassays: RESIST-4 O.K.N.V (Coris BioconceptR) and NG Test Carba 5R (NG BiotechR). Results. In total, 76 KPC, 7 VIM, 1 NDM, 1 OXA-48 and 1 isolate coproducing KPC + NDM enzymes were included. The concordance of different methods estimated by Kappa index was 0.432 (Standard error: 0.117), thus showing a high variability with the synergy tests with boronic acid and EDTA and reporting 16 false negatives that were detected by the two immunochromatographic methods. Co-production was only detected using immunoassays. Conclusion. Conventional phenotypic synergy tests with boronic acid and EDTA used for detecting carbapenemases are suboptimal and their routine use should be reconsidered. They depend on the degree of enzyme expression and the distance between disks. Lateral flow immunoassay tests are a rapid and cost-effective tool to detect and differentiate carbapenemases, improving clinical outcomes through targeted therapy and promoting infection prevention measures.
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Background: Atypical Hemolytic Uremic Syndrome (aHUS) is a complement-mediated thrombotic microangiopathy. Pathophysiological mechanism involves uncontrolled complement activation due to a genetic or acquired anomaly coupled with a triggering event. We report a case of aHUS recurrence following COVID-19 vaccination. Material and methods: Whole blood (EDTA) was collected and processed with CD46-PE, CD45-PerCP, isotype control-PE markers. Staining was measured through median fluorescence intensity and expressed as CD46/isotype ratio. Sanger sequencing was used for identification of variants in CD46 gene. All the participants provided informed written consent. Results: Proband (P) is a 39-year-old woman admitted for nausea, vomiting, epigastric pain and haematuria, three days after first dose of ChAdOx1 nCov-19 vaccine. Laboratory testing showed MAHA (Hb:8.8g/dL, Ht:26%), thrombocytopenia (80x109/mm3) and acute kidney injury (Cr:2.15mg/dL, Ur:92mg/dL). P and three of her siblings have experienced recurrent TMA episodes since childhood. In 2019, genetic study from P's sister (S) identified two heterozygous variants in CD46, one pathogenic (Glu179Gln) and one of uncertain significance (Cys94Tyr). We demonstrated that P carries the same variants and observed a 50% decrease of CD46 expression in both P and S (fig.1). Platelet transfusion, corticosteroids and 9 sessions of plasmapheresis contributed to rapid recovery of P. Discussion: Glu179Gln was reported to increase CD46 expression on granulocytes in aHUS patient and to reduce C4b cofactor activity1. We observed that combination of Glu179Gln and Cys94Tyr was associated with low levels of CD46 on cell surface. Conclusion: This case report supports the evidence of COVID-19 vaccine as a precipitating event for aHUS recurrence.
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Introduction: India is world’s second most populous country with tropical climate and highly dense overcrowded areas which make it an easy target for spread of covid infection. As a preventive measure the government of India launched a vaccination drive from 16th January 2021, currently as of September 2021 more than 806 million doses of the approved vaccine- (covishield- astra Zeneca and covaxin- bharat biotech) have been given. They are both adenovirus carrying viral load and inactivated vaccine respectively. Methods: We will now describe a series of 7 cases of immunogenic glomerulonephritis response following covid vaccination which have been admitted in our tertiary care hospital in southern India between a period of January 2021- September 2021 with signs and symptoms of any of the following nephrotic/ nephritic syndrome, rapidly progressive glomerular disease, isolated proteinurea, hematuria, acute kidney injury and thrombotic complications. All these patients had a similarity in common- all had received single/ 2nd dose of covid vaccine in the last 15 days before the onset of symptoms. All Patients underwent renal biopsy for histological confirmation of the diagnosis as well as covid antibody level testing to see for any relation between the two. once diagnoses was confirmed patients were started on immunosupression in form of steroids, plasmapheresis and other supportive measures like ACE/ ARBs.their outcomes were recorded serially as to whether complete/ partial or no remission was achieved. Results: [Formula presented] All characteristics of the patients including the baseline characteristics- complete blood picture, serum creatinine, electrolytes, 24 hour urine proteinurea. The vaccine type and duration of appearance of symptoms,the renal biopsy findings, the management of each patient and their outcome have been described in the table attached. we had case series of 7 patients with different presentation of glomerulonephritis in the past 9 months with a history of temporal association with covid vaccination in last fortnight. Conclusions: The Immunonephrology Working Group of the ERA-EDTA recently published recommendations on the use of COVID-19 vaccines in patients with autoimmune kidney diseases and supports the vaccination of all individuals without known contraindications. However, these recommendations did not advise on whether vaccination with one vaccine platform was preferable to another. Vigilance should be exercised in patients presenting with new-onset urinary abnormalities and hypertension following COVID-19 vaccination.Besides urinary tract infection and urological causes,glomerulonephritis should be considered in patients with non-resolving macroscopic hematuria. Proteinurea and active sediments in urine. Meanwhile, these isolated reports should not lead to vaccine hesitation during this pandemic as the benefits of vaccination strongly outweigh potential risks. No conflict of interest
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Introduction: COVID-19 caused by SARS-CoV-2is a highly contagious disease. It is a complex systemic disease primarily involving therespiratory system. Patients usually have mild to moderate illness andpresent with flu like symptoms. However, a small group of patientsmay progress or present in a critical condition necessitating intensivecare. Early identification of risk factors associated with critical illnessmay aid in providing timely supportive care and access to theintensive care unit (ICU) when required.Aims &Objectives: To compare the CBC findings, NeutrophilLymphocyte Ratio (NLR), derived neutrophil lymphocyte ratio (dNLR), Platelet Lymphocyte Ratio (PLR) and Lymphocyte MonocyteRatio (LMR) in mild and severe categories of COVID 19 patients.Materials &Methods: Cases diagnosed as COVID 19 and admittedin the wards and ICU of GTB Hospital, Delhi were included. Sampleswere collected from 27 cases. The cases were divided in severe andmild categories. Blood was collected in EDTA vial and CBC wasdone using Automated Hematology Analyser. Data were entered inSPSS 26 and statistical analysis was done using same software.Result: NLR, dNLR values and TLC were found to be significantlyhigher (P< 0.05) in severe COVID-19 patients compared to mildCOVID-19 patients. Differences in platelet count, PLR and LMRvalues were not significant between the two groups. Using ROCcurve, a cut off value of 10,950 for TLC, 6.19 for NLR and 4.13 fordNLR were determined. Using these values TLC was found to be93.3% sensitive, 91.7% specific;NLR was found to be 73.3% sensitive, 72.7% specific;dNLR was found to be 80% sensitive, 81.8%specific in recognizing severe COVID 19 infections.Conclusions: NLR, d-NLR, PLR &LMR are readily accessiblebiomarker, which can be calculated based on a complete blood count.In our study, TLC, NLR and dNLR were found to be useful indetecting severe COVID 19 infections. TLC was the most sensitiveand specific marker in detecting severe COVID 19 patients followedby dNLR. NLR was less valuable than dNLR.
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Introduction: Mechanisms underlying allergic reactions to the new BNT162b2 (Pfizer) and AZD1222 (AstraZeneca) COVID-19 vaccinations are poorly understood. Polyethylene glycol (PEG) is implicated for BNT162b2;and polysorbate 80 (PS80) and disodium edetate (EDTA) for AZD1222. Methods: Patients referred to our service were investigated with standardised vaccine skin-prick testing (SPT) and intradermal testing (IDT) protocols. Basophil activation testing (BAT) was performed in patients with history highly suggestive of excipient or vaccine allergy. Results: Reason for referral was suspected excipient allergy in 16/23 (70%), previous other vaccine reaction in 4/23 (17%), and reaction to the BNT162b2 vaccine in 3/23 (13%). In patients with suspected excipient allergy, SPT was only positive in 1/16 (6%). In 12/16 patients with suspected PEG allergy, IDT and BAT were positive in 5 (42%) for the BNT162b2 vaccine but not PEG. 3/5 have subsequently undergone successful vaccination with AZD1222, while 1/5 had cross-reactivity with AZD1222 on BAT and has not been vaccinated. 2/16 patients with suspected PS80 allergy were negative on SPT, IDT, and BAT, and have undergone successful AZD1222 vaccination. In the 2/16 patients with EDTA allergy IDT was positive to EDTA but neither vaccine, correlating with BAT. 1 has been successfully vaccinated with the EDTA-containing AZD1222 vaccine. 2 patients (1 reaction to BNT162b2, 1 other vaccine reaction) developed systemic reactions during testing without tryptase elevation. Both were associated with local flare response to the BNT162b2 vaccine, both have undergone successful vaccination with the AZD1222 vaccine. All other patients with negative SPT, IDT, and BAT results have subsequently tolerated vaccination. Conclusion: Most patients can be successfully vaccinated with available COVID-19 vaccines. SPT has low sensitivity and a combined protocol of SPT, IDT, and BAT provides confidence in allergy delabelling. Not all excipient allergies correlate to vaccination allergy and BAT provides a powerful diagnostic tool in these cases.
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[Formula presented] Background and Objective The rapid spread of the COVID -19 pandemic and the high variability of the course of the disease make it essential to search for early predictors of outcome. The objective of our study is to predict severe SARS COV2 pneumonia using early cytometric profiles Material and Methods Prospective and observational study of adults with confirmed COVID-19 infection admitted on Emergency Department (ED). We collected epidemiological, clinical and laboratory data of every patient until they were discharged or died. Multiparametric flow cytometry (FC) analysis of T-lymphocytes (CD4, CD8, CD4 activated, CD8 activated, naïve (Tn), central-memory (Tcm), effector-memory (Tem), effector (Te) and Th17 subsets), B-lymphocytes (naïve, memory, transitional subsets, and assessment of clonality), NK cells, plasmablasts, p-DCs (plasmacytoid dendritic cells), m-DCs (myeloid dendritic cells), basophils, and monocytes (MO1, MO2, MO3, slan+ MO3) was performed on whole peripheral blood collected on EDTA, before immunosuppressive therapy was started. We designed a 7-tube 8-color experimental panel. Cell surface staining of 2 × 106 cells was performed and at least 500 000 total events were acquired for the assessment of plasmablasts, p-DCs, m-DCs, basophils, and the monocyte subsets;for the study of B, T and NK-lymphocyte populations we acquired at least 100 000 total events (FACS Canto II;BD Biosciences). Severity was assessed on the basis of World Health Organization´s (WHO) international 10 level ordinal scale (WHOs) and also according to 4 respiratory status, based on SpO2(peripheral blood oxygen saturation)/FIO2 (fraction of inspired oxygen) ratio (SpFi). SpFi group 1 >452, SpFi2 2: 315-452;SpFi 3 236-315, SpFi 4: <236 (respiratory distress) Results 53 patients were included: epidemiological and clinical data available on table 1. 23 patients (43.39%) arrived to SpFi4 status. WHOs >6 (WHO 6:oxygen by non invasive ventilation or high flow) was achieved by 20 patients (37.7%) Good prognosis (meaning SpFi1 as the worse respiratory status in the follow -up) was associated to cytometric profiles: there was a significant increase in CD3, p-DCs, m-DCs, basophils, monocytes, Tcm, % of lymphocytes and CD3/CD19 ratio whereas there was a significant reduction in CD19, % of neutrophils and % Neutrophils/% lymphocyte ratio. In the SpFi4 group, there was a significant reduction of CD3, p-DCs, m-DCs, plasmablasts and CD3/NK ratio. In patients starting in SpFi group 1-2 in the ED but progressing to SpFi 3-4 during the follow up (27 patients), there was a statistically significant relation with Tn, Te and Tn/Te ratio (Tn/Te ratio <0.717: OR 13.5 (p 0.002, [95% CI 2.552-71.403). Initial SpFi1 patients that evolved to SpFi 3-4 during follow up (10 patients), presented a Tn/Te ratio < 0.717 with an OR 11.556 (p =0.005, [95% CI 2.059-64.853]). Plasmablasts < 0.075 and CD3/NK <5.71, were identified as independent risk factors for SpFI4 during follow up. After multivariable analysis, both variables kept their significance: CD3/NK (OR 11,247, p=0.005) and plasmablasts (OR 12,524, p=0.004). About prediction of WHOs >6, multivariable analysis showed CD3/NK <5.71 (OR 22,240 [95%CI 2,340-211,342] p= 0.007) and plasmablasts<0.075 (OR 28.635 [95% CI 3,187-257,301] p=0.003). A score (0,1,2) comprising both risk factors, was significantly predictive of SpFI4, regardless of the initial respiratory status, age or days from symptoms onset. In our cohort, only 1/15 (6.7%) patients with 0 points (neither plasmablasts nor CD3/NK score), arrived to SpFi4. However, 10/11 (90.9%) patients with 2 points, reached to SpFi4 respiratory status (C-index = 0.837) Same score was applied to predict WHOs > 6: 90.9% with 2 points progressed to WHO>6 and 0/15 patients with 0 points reached the same goal (C-index = 0.872) An incidental finding of 4 indolent B-lymphoproliferative disorders (2CLL-like MBLs and 2non -CLL-like MBL), was found, and they were associated with older age and progression to death. Conclusions Flow cytometry on whole pe ipheral blood samples of SARS-COV2 pneumonia patients, collected before corticosteroid or immunosuppressive therapy, could identify cytometric patterns associated to prognosis. Plasmablasts, mDCs and pDCs levels as well as CD3/NK ratio, are associated to a worse respiratory status, while Tn/Te ratio could detect non-severe patients who will require high-flow oxygen devices during follow up. [Formula presented] Disclosures: No relevant conflicts of interest to declare.