LONGITUDINAL CLUSTER ANALYSIS OF HEMODIALYSIS PATIENTS WITH COVID-19 IN THE PRE-VACCINATION ERA

Coronavirus disease 2019 (COVID-19) is characterized by a high heterogeneity of clinical presentation and outcomes. This is also true for patients undergoing maintenance hemodialysis (HD), who, due to specific clinical factors and immune status, represent a distinct subgroup of COVID-19 patients. Starting from this observation in this research letter we tested and validated in two cohorts of HD patients with COVID-19 (derivation and validation cohort, respectively) an innovative model which combines linear mixed effect modeling and cluster analysis on longitudinal. This study aimed to describe a methodology allowing patient stratification from simple and widely available data. Our results could be interesting not only to improve COVID-19 management but also to support the application of longitudinal cluster analysis strategy in other clinical settings.

An optimized flow cytometry protocol for simultaneous detection of T cell activation induced markers and intracellular cytokines: application to SARS-Cov-2 vaccinated individuals

Antigen (ag)-specific T cell analysis is an important step for investigation of cellular immunity in many settings, such as infectious diseases, cancer and vaccines. Multiparameter flow cytometry has advantages in studying both the rarity and heterogeneity of these cells. In the cellular immunologists toolbox, the expression of activation-induced markers (AIM) following antigen exposure has made possible the study and sorting of ag-specific T cells without using human leukocyte antigen (HLA)-multimers. In parallel, assessing the cytokine profile of responding T cells would support a more comprehensive description of the ongoing immune response. Here, a method and flow cytometry panel were optimized to combine the detection of activated CD4+ and CD8+ T cells in a TCR-dependent manner with the evaluation of cytokine production by intracellular staining, without affecting the positivity of activation markers. In particular, the expression of CD134 (OX40) and CD69 have been tested in conjunction with intracellular (ic) CD137 (4-1BB) to detect SARS-Cov-2 Spike protein-specific activated T cells. In our setting, assessing CD134 provided minimal contribution to detect the pool of AIM+ T cells, whereas a key role was described for ic-CD69, which was co-expressed with ic-CD137 in both CD4+ and CD8+ lymphocytes. Moreover, the analysis of TCR-triggered cytokine-producing T cells (IFN{gamma}, TNF and IL-2 were assessed) further confirmed the capacity of ic-CD69 to identify functionally responsive antigen-specific T cells, which were often largely negative or poorly positive for CD134 expression. In parallel, the use of CD45RA, CCR7 and CXCR5 allowed us to describe the T cell matuarion curve and detect T helper follicular CD4+ cells, including the antigen-specific activated subsets. In conclusion, we optimized a method and flow cytometry panel combining assessment of activation induced markers and intracellular cytokines that will be useful for measuring TCR stimulation-dependent activation of CD4+ and CD8+ T cells.

The longest persistence of viable SARS-CoV-2 with recurrence of viremia and relapsing symptomatic COVID-19 in an immunocompromised patient - a case study

BackgroundImmunocompromised patients show prolonged shedding of SARS-CoV-2 in nasopharyngeal swabs. We report a case of a prolonged persistence of viable SARS-CoV-2 associated with clinical relapses of COVID-19 in a lymphoma patient. MethodsNasopharyngeal swabs and blood samples were tested for SARS-CoV-2 by Real time-PCR (RT-PCR). On five positive nasopharyngeal swabs, we performed viral culture and next generation sequencing. We analysed the patients adaptive and innate immunity to characterize T and NK cell subsets. FindingsSARS-CoV-2 RT-PCR on nasopharyngeal swabs samples remained positive with cycle threshold mean values of 22 {+/-} 1{middle dot}3 for over 8 months. All five performed viral cultures were positive and genomic analysis confirmed a persistent infection with the same strain. Viremia resulted positive in three out of four COVID-19 clinical relapses and cleared each time after remdesivir treatment. T and NK cells dynamic was different in aviremic and viremic samples and no SARS-CoV-2 specific antibodies were detected throughout the disease course. InterpretationIn our patient, SARS-CoV-2 persisted with proven infectivity for over eight months. Viremia was associated with COVID-19 relapses and remdesivir treatment was effective in viremia clearance and symptoms remission, although it was unable to clear the virus from the upper respiratory airways. During the viremic phase, we observed a low frequency of terminal effector CD8+ T lymphocytes in peripheral blood that are probably recruited in inflammatory tissue for viral eradication. In addition we found a high level of NK cells repertoire perturbation with a relevant involvement during SARS-CoV-2 viremia. FundingNone.