Epigenetic immune monitoring for COVID-19 disease course prognosis.

Background: The course of COVID-19 is associated with severe dysbalance of the immune system, causing both leukocytosis and lymphopenia. Immune cell monitoring may be a powerful tool to prognosticate disease outcome. However, SARS-CoV-2 positive subjects are isolated upon initial diagnosis, thus barring standard immune monitoring using fresh blood. This dilemma may be solved by epigenetic immune cell counting. Methods: In this study, we used epigenetic immune cell counting by qPCR as an alternative way of quantitative immune monitoring for venous blood, capillary blood dried on filter paper (dried blood spots, DBS) and nasopharyngeal swabs, potentially allowing a home-based monitoring approach. Results: Epigenetic immune cell counting in venous blood showed equivalence with dried blood spots and with flow cytometrically determined cell counts of venous blood in healthy subjects. In venous blood, we detected relative lymphopenia, neutrophilia, and a decreased lymphocyte-to-neutrophil ratio for COVID-19 patients (n =103) when compared with healthy donors (n = 113). Along with reported sex-related differences in survival we observed dramatically lower regulatory T cell counts in male patients. In nasopharyngeal swabs, T and B cell counts were significantly lower in patients compared to healthy subjects, mirroring the lymphopenia in blood. Naïve B cell frequency was lower in severely ill patients than in patients with milder stages. Conclusions: Overall, the analysis of immune cell counts is a strong predictor of clinical disease course and the use of epigenetic immune cell counting by qPCR may provide a tool that can be used even for home-isolated patients.

Operational considerations for respiratory virus surveillance in Europe, 18 July 2022

This document outlines operational considerations to support the continuity of national surveillance systems and public health laboratories for epidemiological and virological surveillance for influenza, SARS-CoV-2, and potentially other respiratory viruses (such as RSV or new viruses of public health concern) in the 2022/2023 winter season and beyond. It builds on previously published documents on COVID-19 and influenza surveillance. The intended audience for this document is those with national responsibility for influenza and COVID-19 surveillance in Europe.

Third dose of anti-SARS-CoV-2 vaccine for patients with cancer: Should humoral responses be monitored? A position article.

Taking into account higher risk of severe coronavirus disease 2019 or death among patients with cancer, as well as impaired immunogenicity after anti-SARS-CoV-2 vaccines, in addition to waning immunity, booster dosing appears mandatory in this patient population. This review sought to provide reasonable evidence so as to assist oncologists in their daily practice, helping them decide when an anti-SARS-Cov2 antibody (Ab) dosage should be scheduled after a full two-dose vaccination and, if necessary, propose an early third dose (D3). Such D3 could apply to non-responder patients with anti-Spike (S) Abs titres <40 binding Ab unit (BAU)/mL. For lowresponder patients with anti-S Ab titres between 40 BAU/mL and 100/260 BAU/mL (suggested area of uncertainty), an early D3 may similarly be proposed. Nevertheless, this D3 could be administered in a less urgent manner, taking into account associated comorbidities and regional epidemic incidence rates. This latter strategy may comprise a monthly dosage of anti-S titres so as to better assess the kinetics of waning immunity. For responder patients with anti-S titres above 260 BAU/mL, we suggest to follow the recommendations outlined for the general population. Given this context, patients with anti-S titres above 1000 BAU/mL should be given the possibility to undergo anti-S titre control after three months, designed to assess rapid humoral waning immunity. We strongly recommend that patients with cancer be included into observational serological monitoring studies or clinical trials that are dedicated to severe immunocompromised patients without any humoral seroconversion after D3.

COVID-19 in Elderly, Immunocompromised or Diabetic Patients-From Immune Monitoring to Clinical Management in the Hospital.

The novel, highly transmissible severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has triggered a pandemic of acute respiratory illness worldwide and remains a huge threat to the healthcare system's capacity to respond to COVID-19. Elderly and immunocompromised patients are at increased risk for a severe course of COVID-19. These high-risk groups have been identified as developing diminished humoral and cellular immune responses. Notably, SARS-CoV-2 RNA remains detectable in nasopharyngeal swabs of these patients for a prolonged period of time. These factors complicate the clinical management of these vulnerable patient groups. To date, there are no well-defined guidelines for an appropriate duration of isolation for elderly and immunocompromised patients, especially in hospitals or nursing homes. The aim of the present study was to characterize at-risk patient cohorts capable of producing a replication-competent virus over an extended period after symptomatic COVID-19, and to investigate the humoral and cellular immune responses and infectivity to provide a better basis for future clinical management. In our cohort, the rate of positive viral cultures and the sensitivity of SARS-CoV-2 antigen tests correlated with higher viral loads. Elderly patients and patients with diabetes mellitus had adequate cellular and humoral immune responses to SARS-CoV-2 infection, while immunocompromised patients had reduced humoral and cellular immune responses. Our patient cohort was hospitalized for longer compared with previously published cohorts. Longer hospitalization was associated with a high number of nosocomial infections, representing a potential hazard for additional complications to patients. Most importantly, regardless of positive SARS-CoV-2 RNA detection, no virus was culturable beyond a cycle threshold (ct) value of 33 in the majority of samples. Our data clearly indicate that elderly and diabetic patients develop a robust immune response to SARS-CoV-2 and may be safely de-isolated at a ct value of more than 35.

Rapid qualitative research to increase COVID-19 vaccination uptake: a research and intervention tool

This qualitative tool provides step by step guidance on how to design and conduct rapid qualitative research with different target groups to understand their barriers and drivers to COVID-19 vaccination. These insights can then be used to inform the development of interventions tailored to the target groups for successful COVID-19 vaccination uptake. Whilst the document focuses on COVID-19 vaccination, the described rapid approach can be used for any vaccination programme.

Early use of canakinumab to prevent mechanical ventilation in select COVID-19 patients: A retrospective, observational analysis.

INTRODUCTION: The fully-human monoclonal anti-interleukin (IL)-1ß antibody canakinumab may inhibit the production of inflammatory mediators in patients with coronavirus disease 2019 (COVID-19) and the hyperinflammatory response potentially leading to acute respiratory distress syndrome. OBJECTIVES: The goal of our retrospective, observational analysis was to evaluate the safety and efficacy of subcutaneous (s.c.) canakinumab in combination with our standard of care (SOC) treatment of selected patients with COVID-19 with respiratory failure and elevated reactive pro-inflammatory markers. METHODS: Eight participants received two doses of s.c. canakinumab 150 mg (or 2 mg/kg for participants weighing ≤40 kg) in addition to SOC. 12 patients received only SOC treatment. RESULTS: Canakinumab treatment reduced the need for mechanical ventilation and reduced proinflammatory markers, resulting in an amelioration of the final outcome, with respect to the control group who received SOC alone. The treatment was safe and well tolerated; no adverse events were reported. CONCLUSION: The use of canakinumab (300 mg, s.c.) in the early stage of COVID-19 with mild-to-moderate respiratory failure was superior to SOC at preventing clinical deterioration and may warrant further investigation as a treatment option for patients with COVID-19 who experience a hyperinflammatory response in the early stage of the disease.

A digital single-molecule nanopillar SERS platform for predicting and monitoring immune toxicities in immunotherapy.

The introduction of immune checkpoint inhibitors has demonstrated significant improvements in survival for subsets of cancer patients. However, they carry significant and sometimes life-threatening toxicities. Prompt prediction and monitoring of immune toxicities have the potential to maximise the benefits of immune checkpoint therapy. Herein, we develop a digital nanopillar SERS platform that achieves real-time single cytokine counting and enables dynamic tracking of immune toxicities in cancer patients receiving immune checkpoint inhibitor treatment - broader applications are anticipated in other disease indications. By analysing four prospective cytokine biomarkers that initiate inflammatory responses, the digital nanopillar SERS assay achieves both highly specific and highly sensitive cytokine detection down to attomolar level. Significantly, we report the capability of the assay to longitudinally monitor 10 melanoma patients during immune inhibitor blockade treatment. Here, we show that elevated cytokine concentrations predict for higher risk of developing severe immune toxicities in our pilot cohort of patients.

The role of PD-L1 in the immune dysfunction that mediates hypoxia-induced multiple organ injury.

Hypoxia is a pathological condition common to many diseases, although multiple organ injuries induced by hypoxia are often overlooked. There is increasing evidence to suggest that the hypoxic environment may activate innate immune cells and suppress adaptive immunity, further stimulating inflammation and inhibiting immunosurveillance. We found that dysfunctional immune regulation may aggravate hypoxia-induced tissue damage and contribute to secondary injury. Among the diverse mechanisms of hypoxia-induced immune dysfunction identified to date, the role of programmed death-ligand 1 (PD-L1) has recently attracted much attention. Besides leading to tumour immune evasion, PD-L1 has also been found to participate in the progression of the immune dysfunction which mediates hypoxia-induced multiple organ injury. In this review, we aimed to summarise the role of immune dysfunction in hypoxia-induced multiple organ injury, the effects of hypoxia on the cellular expression of PD-L1, and the effects of upregulated PD-L1 expression on immune regulation. Furthermore, we summarise the latest information pertaining to the involvement, diagnostic value, and therapeutic potential of immunosuppression induced by PD-L1 in various types of hypoxia-related diseases, including cancers, ischemic stroke, acute kidney injury, and obstructive sleep apnoea. Video Abstract.

Impaired humoral and cellular immunity after SARS-CoV-2 BNT162b2 (tozinameran) prime-boost vaccination in kidney transplant recipients.

Novel mRNA-based vaccines have been proven to be powerful tools in combating the global pandemic caused by SARS-CoV-2, with BNT162b2 (trade name: Comirnaty) efficiently protecting individuals from COVID-19 across a broad age range. Still, it remains largely unknown how renal insufficiency and immunosuppressive medication affect development of vaccine-induced immunity. We therefore comprehensively analyzed humoral and cellular responses in kidney transplant recipients after the standard second vaccination dose. As opposed to all healthy vaccinees and the majority of hemodialysis patients, only 4 of 39 and 1 of 39 transplanted individuals showed IgA and IgG seroconversion at day 8 ± 1 after booster immunization, with minor changes until day 23 ± 5, respectively. Although most transplanted patients mounted spike-specific T helper cell responses, frequencies were significantly reduced compared with those in controls and dialysis patients and this was accompanied by a broad impairment in effector cytokine production, memory differentiation, and activation-related signatures. Spike-specific CD8+ T cell responses were less abundant than their CD4+ counterparts in healthy controls and hemodialysis patients and almost undetectable in transplant patients. Promotion of anti-HLA antibodies or acute rejection was not detected after vaccination. In summary, our data strongly suggest revised vaccination approaches in immunosuppressed patients, including individual immune monitoring for protection of this vulnerable group at risk of developing severe COVID-19.

Approach to SARS-CoV-2 Vaccination in Patients With Multiple Sclerosis.

For more than a year now, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been causing the coronavirus disease (COVID-19) pandemic with high mortality and detrimental effects on society, economy, and individual lives. Great hopes are being placed on vaccination as one of the most potent escape strategies from the pandemic and multiple vaccines are already in clinical use. However, there is still a lot of insecurity about the safety and efficacy of vaccines in patients with autoimmune diseases like multiple sclerosis (MS), especially under treatment with immunomodulatory or immunosuppressive drugs. We propose strategic approaches to SARS-CoV-2 vaccination management in MS patients and encourage fellow physicians to measure the immune response in their patients. Notably, both humoral and cellular responses should be considered since the immunological equivalent for protection from SARS-CoV-2 after infection or vaccination still remains undefined and will most likely involve antiviral cellular immunity. It is important to gain insights into the vaccine response of immunocompromised patients in order to be able to deduce sensible strategies for vaccination in the future.

Parameters of immunoglobulin extraction from dried blood spot cards and immunoassays for detection of antibody response to pathogens including the novel SARS-CoV-2.

Dried blood spots (DBS) are routinely used in screening newborns for treatable disorders. Immunoglobulin extraction from DBS, serum or other biological fluids loaded on filter paper cards could represent a valuable method of specimen preservation in monitoring immune response against pathogens as well as vaccination efficiency. In this study using different sources including serum, and monoclonal antibodies we established parameters for antibody extraction from the filter cards to assess antibody reactivity against Helicobacter pylori, measles virus (MV) and the novel coronavirus SARS-CoV-2 antigens. We demonstrated that DBS and dried undiluted serum result in completely preserved antibody activity for immunoassays, including in virus neutralization assays against MV. Extraction efficiency was determined by IgG concentration measurements. The plaque-reduction neutralization titer 50% of dried human serum spots remained stable after more than 10-day storage - 1:359 vs. 1:345 for the corresponding frozen sample. DBSs could be used to monitor immune response to bacterial and viral antigens following natural exposure or immunization. Mice immunized with recombinant spike protein receptor-binding domain of SARS-CoV-2 developed a strong antibody response by day 14 and reached titers above 1:64,000 on day 21 following the secondary boost immunization as measured on DBS samples in antigen-mediated ELISA. Variability in IgG concentration of eluted DBS could be influenced by factors involved in sample application, extraction process and sample characteristics. Adjustment of antibody specific activity to the eluted IgG concentration can increase accuracy of the result interpretation, including in SARS-CoV-2 serological diagnostics.

A quadruple blind, randomised controlled trial of gargling agents in reducing intraoral viral load among hospitalised COVID-19 patients: A structured summary of a study protocol for a randomised controlled trial.

OBJECTIVES: 1- To compare the effectiveness of 1% Hydrogen peroxide, 0.2% Povidone-Iodine, 2% hypertonic saline and a novel solution Neem extract (Azardirachta indica) in reducing intra-oral viral load in COVID-19 positive patients. 2- To determine the salivary cytokine profiles of IL-2, IL-4, IL-6, IL-10, TNF-α, IFN-γ and IL- 17 among COVID-19 patients subjected to 1% Hydrogen peroxide, 0.2% Povidone-Iodine, 2% hypertonic saline or Neem extract (Azardirachta indica) based gargles. TRIAL DESIGN: This will be a parallel group, quadruple blind-randomised controlled pilot trial with an add on laboratory based study. PARTICIPANTS: A non-probability, purposive sampling technique will be followed to identify participants for this study. The clinical trial will be carried out at the Aga Khan University Hospital (AKUH), Karachi, Pakistan. The viral PCR tests will be done at main AKUH clinical laboratories whereas the immunological tests (cytokine analysis) will be done at the Juma research laboratory of AKUH. The inclusion criteria are laboratory-confirmed COVID-19 positive patients, male or female, in the age range of 18-65 years, with mild to moderate disease, already admitted to the AKUH. Subjects with low Glasgow coma score, with a history of radiotherapy or chemotherapy, who are more than 7 days past the onset of COVID- 19 symptoms, or intubated or edentulous patients will be excluded. Patients who are being treated with any form of oral or parenteral antiviral therapy will be excluded, as well as patients with known pre-existing chronic mucosal lesions such as lichen planus. INTERVENTION AND COMPARATOR: Group A (n=10) patients on 10 ml gargle and nasal lavage using 0.2% Povidone-Iodine (Betadiene® by Aviro Health Inc./ Pyodine® by Brooks Pharma Inc.) for 20-30 seconds, thrice daily for 6 days. Group B (n=10) patients will be subjected to 10 ml gargle and nasal lavage using 1% Hydrogen peroxide (HP® by Karachi Chemicals Products Inc./ ActiveOxy® by Boumatic Inc.) for 20-30 seconds, thrice daily for 6 days. Group C will comprised of (n=10) subjects on 10ml gargle and nasal lavage using Neem extract solution (Azardirachta indica) formulated by Karachi University (chemistry department laboratories) for 20-30 seconds, thrice daily for 6 days. Group D (n=10) patients will use 2% hypertonic saline (Plabottle® by Otsuka Inc.) gargle and nasal lavage for a similar time period. Group E (n=10) will serve as positive controls. These will be given simple distilled water gargles and nasal lavage for 20-30 seconds, thrice daily for six days. For nasal lavage, a special douche syringe will be provided to each participant. Its use will be thoroughly explained by the data collection officer. After each use, the patient is asked not to eat, drink, or rinse their mouth for the next 30 minutes. MAIN OUTCOMES: The primary outcome is the reduction in the intra-oral viral load confirmed with real time quantitative PCR. RANDOMISATION: The assignment to the study group/ allocation will be done using the sealed envelope method under the supervision of Clinical Trial Unit (CTU) of Aga Khan University, Karachi, Pakistan. The patients will be randomised to their respective study group (1:1:1:1:1 allocation ratio) immediately after the eligibility assessment and consent administration is done. BLINDING (MASKING): The study will be quadruple-blinded. Patients, intervention provider, outcome assessor and the data collection officer will be blinded. The groups will be labelled as A, B, C, D or E. The codes of the intervention will be kept in lock & key at the CTU and will only be revealed at the end of study or if the study is terminated prematurely. NUMBERS TO BE RANDOMISED (SAMPLE SIZE): As there is no prior work on this research question, so no assumptions for the sample size calculation could be made. The present study will serve as a pilot trial. We intend to study 50 patients in five study groups with 10 patients in each study group. For details, please refer to Fig. 1 for details. TRIAL STATUS: Protocol version is 7.0, approved by the department and institutional ethics committees and clinical trial unit of the university hospital. Recruitment is planned to start as soon as the funding is sanctioned. The total duration of the study is expected to be 6 months i.e. August 2020-January 2021. TRIAL REGISTRATION: This study protocol was registered at www.clinicaltrials.gov on 10 April 2020 NCT04341688 . FULL PROTOCOL: The full protocol is attached as an additional file, accessible from the Trials website (Additional file 1). In the interest in expediting dissemination of this material, the familiar formatting has been eliminated; this Letter serves as a summary of the key elements of the full protocol. The study protocol has been reported in accordance with the Standard Protocol Items: Recommendations for Clinical Interventional Trials (SPIRIT) guidelines (Additional file 2). Fig. 1 Flow diagram of study-participants' timeline.

Cancer, COVID-19, and Antiviral Immunity: The CAPTURE Study.

The SARS-CoV-2 pandemic has posed a significant challenge for risk evaluation and mitigation among cancer patients. Susceptibility to and severity of COVID-19 in cancer patients has not been studied in a prospective and broadly applicable manner. CAPTURE is a pan-cancer, longitudinal immune profiling study designed to address this knowledge gap.

Anti SARS-CoV-2 antibodies monitoring in a group of residents in a long term care facility during COVID-19 pandemic peak.

Objectives Clinical laboratories plays a key role in screening, diagnosis and containment of the Coronavirus 2019 infection epidemic. The etiological diagnosis presupposes the isolation of virus genetic material in the patient's biological sample but laboratory diagnostics also make use of searching possibility for immunoglobulin (Ig)G, IgM classes antibodies. The characteristics of the antibody response are not yet completely clear. Methods This study describes a serological monitoring of subjects, elderly nursing care residence guests, interested by a very large infection outbreak. After first nasopharyngeal swab, all the positive subjects (43) were monitored for the persistence of the virus infection through nasopharyngeal swab after 20 days (16-24), 32 days (28-36) and after 49 days (47-50). At the same time, during the second (day 32) and third (day 49) follow up, all the guests were investigated for IgM and IgG anti SARS-CoV-2 antibodies, by using a quantitative chemiluminescence method. Results Thirty two days after performing the first diagnostic swab, 39 of 43 patients (90%) had IgG higher than the cut off value. After 49 days the four patients with negative IgG were still negative. The comparison of the levels of IgG-Ab between the controls shows a significant decrease in concentrations (-10%). Conclusions Our study confirms that in most patients affected by COVID-19 there is a typical antibody response with IgG-Ab present in 90% of nursing care COVID-19 positive residence guests. For IgM-Ab only 23% of tested subjects were positive on the 32nd and 49th day of illness, always in parallel with the IgG-Ab positivity.

Clinical benefits of Tocilizumab in COVID-19-related cytokine release syndrome in a patient with end-stage kidney disease on haemodialysis in Australia.

COVID-19 remains a global pandemic with more than 10 million cases and half a million deaths worldwide. The disease manifestations in patients with chronic kidney disease and especially those on haemodialysis are still being understood, with only a few overseas case series, and small observational trials thus far. It appears that the disease is more severe in this patient cohort. Part of the pathophysiology of severe COVID-19 is related to accompanying cytokine release syndrome (CRS). Tocilizumab, an interleukin-6 inhibitor, has been trialled for treatment of CRS in COVID-19, but not yet approved. We present a case of an Australian patient on long-term haemodialysis with severe COVID-19 who was successfully treated with Tocilizumab. The peak of her illness was on day 7, with a C-reactive protein of 624 mg/L (reference < 5 mg/L), ferritin of 5293 ng/mL (reference 30-500 ng/mL), and interleukin-6 level 1959.7 pg/mL, consistent with CRS. She was severely hypoxic on a ventilator, with rising inotropic requirements. With the use of Tocilizumab, there was a significant and immediate response in her inflammatory markers, and she made a steady recovery. The patient was discharged home 6 weeks after presentation.

Immune monitoring facilitates the clinical decision in multifocal COVID-19 of a pancreas-kidney transplant patient.

The optimal management in transplant recipients with coronavirus disease 2019 (COVID-19) remains uncertain. The main concern is the ability of immunosuppressed patients to generate sufficient immunity for antiviral protection. Here, we report on immune monitoring facilitating a successful outcome of severe severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-associated pneumonia, meningoencephalitis, gastroenteritis, and acute kidney and pancreas graft failure in a pancreas-kidney transplant recipient. Despite the very low numbers of circulating B, NK, and T cells identified in follow-up, a strong SARS-CoV-2 reactive T cell response was observed. Importantly, we detected T cells reactive to Spike, Membrane, and Nucleocapsid proteins of SARS-CoV-2 with majority of T cells showing polyfunctional proinflammatory Th1 phenotype at all analyzed time points. Antibodies against Spike protein were also detected with increasing titers in follow-up. Neutralization tests confirmed their antiviral protection. A correlation between cellular and humoral immunity was observed underscoring the specificity of demonstrated data. We conclude that analyzing the kinetics of nonspecific and SARS-CoV-2-reactive cellular and humoral immunity can facilitate the clinical decision on immunosuppression adjustment and allow successful outcome as demonstrated in the current clinical case. Although the antiviral protection of the detected SARS-CoV-2-reactive T cells requires further evaluation, our data prove an ability mounting a strong SARS-CoV-2-reactive T cell response with functional capacity in immunosuppressed patients.

Clinical study using mesenchymal stem cells for the treatment of patients with severe COVID-19.

The Coronavirus disease 2019 (COVID-19) caused by SARS-CoV-2 was identified in December 2019. The symptoms include fever, cough, dyspnea, early symptom of sputum, and acute respiratory distress syndrome (ARDS). Mesenchymal stem cell (MSC) therapy is the immediate treatment used for patients with severe cases of COVID-19. Herein, we describe two confirmed cases of COVID-19 in Wuhan to explore the role of MSC in the treatment of COVID-19. MSC transplantation increases the immune indicators (including CD4 and lymphocytes) and decreases the inflammation indicators (interleukin-6 and C-reactive protein). High-flow nasal cannula can be used as an initial support strategy for patients with ARDS. With MSC transplantation, the fraction of inspired O2 (FiO2) of the two patients gradually decreased while the oxygen saturation (SaO2) and partial pressure of oxygen (PO2) improved. Additionally, the patients' chest computed tomography showed that bilateral lung exudate lesions were adsorbed after MSC infusion. Results indicated that MSC transplantation provides clinical data on the treatment of COVID-19 and may serve as an alternative method for treating COVID-19, particularly in patients with ARDS.