How Yeast Antifungal Resistance Gene Analysis Is Essential to Validate Antifungal Susceptibility Testing Systems.

The antifungal susceptibility testing (AFST) of yeast pathogen alerts clinicians about the potential emergence of resistance. In this study, we compared two commercial microdilution AFST methods: Sensititre YeastOne read visually (YO) and MICRONAUT-AM read visually (MN) or spectrophotometrically (MNV), interpreted with Clinical and Laboratory Standards Institute and European Committee on Antimicrobial Susceptibility Testing criteria, respectively. Overall, 97 strains from 19 yeast species were measured for nine antifungal drugs including a total of 873 observations. First, the minimal inhibitory concentration (MIC) was compared between YO and MNV, and between MNV and MN, either directly or by assigning them to five susceptibility categories. Those categories were based on the number of MIC dilutions around the breakpoint or epidemiological cut-off reference values (ECOFFs or ECVs). Second, YO and MNV methods were evaluated for their ability to detect the elevation of MICs due to mutation in antifungal resistance genes, thanks to pairs or triplets of isogenic strains isolated from a single patient along a treatment previously analyzed for antifungal resistance gene mutations. Reproducibility measurement was evaluated, thanks to three quality control (QC) strains. YO and MNV direct MIC comparisons obtained a global agreement of 67%. Performing susceptibility category comparisons, only 22% and 49% of the MICs could be assigned to categories using breakpoints and ECOFFs/ECVs, respectively, and 40% could not be assigned due to the lack of criteria in both consortia. The YO and MN susceptibility categories gave accuracies as low as 50%, revealing the difficulty to implement this method of comparison. In contrast, using the antifungal resistance gene sequences as a gold standard, we demonstrated that both methods (YO and MN) were equally able to detect the acquisition of resistance in the Candida strains, even if MN showed a global lower MIC elevation than YO. Finally, no major differences in reproducibility were observed between the three AFST methods. This study demonstrates the valuable use of both commercial microdilution AFST methods to detect antifungal resistance due to point mutations in antifungal resistance genes. We highlighted the difficulty to conduct conclusive analyses without antifungal gene sequence data as a gold standard. Indeed, MIC comparisons taking into account the consortia criteria of interpretation remain difficult even after the effort of harmonization

Novel birch pollen specific immunotherapy formulation based on contiguous overlapping peptides.

BACKGROUND: Synthetic contiguous overlapping peptides (COPs) may represent an alternative to allergen extracts or recombinant allergens for allergen specific immunotherapy. In combination, COPs encompass the entire allergen sequence, providing all potential T cell epitopes, while preventing IgE conformational epitopes of the native allergen. METHODS: Individual COPs were derived from the sequence of Bet v 1, the major allergen of birch pollen, and its known crystal structure, and designed to avoid IgE binding. Three sets of COPs were tested in vitro in competition ELISA and basophil degranulation assays. Their in vivo reactivity was determined by intraperitoneal challenge in rBet v 1 sensitized mice as well as by skin prick tests in volunteers with allergic rhinoconjunctivitis to birch pollen. RESULTS: The combination, named AllerT, of three COPs selected for undetectable IgE binding in competition assays and for the absence of basophil activation in vitro was unable to induce anaphylaxis in sensitized mice in contrast to rBet v 1. In addition no positive reactivity to AllerT was observed in skin prick tests in human volunteers allergic to birch pollen. In contrast, a second set of COPs, AllerT4-T5 displayed some residual IgE binding in competition ELISA and a weak subliminal reactivity to skin prick testing. CONCLUSIONS: The hypoallergenicity of contiguous overlapping peptides was confirmed by low, if any, IgE binding activity in vitro, by the absence of basophil activation and the absence of in vivo induction of allergic reactions in mouse and human. TRIAL REGISTRATION: ClinicalTrials.gov NCT01719133

Heterogenous humoral and cellular immune responses with distinct trajectories post-SARS-CoV-2 infection in a population-based cohort.

To better understand the development of SARS-CoV-2-specific immunity over time, a detailed evaluation of humoral and cellular responses is required. Here, we characterize anti-Spike (S) IgA and IgG in a representative population-based cohort of 431 SARS-CoV-2-infected individuals up to 217 days after diagnosis, demonstrating that 85% develop and maintain anti-S responses. In a subsample of 64 participants, we further assess anti-Nucleocapsid (N) IgG, neutralizing antibody activity, and T cell responses to Membrane (M), N, and S proteins. In contrast to S-specific antibody responses, anti-N IgG levels decline substantially over time and neutralizing activity toward Delta and Omicron variants is low to non-existent within just weeks of Wildtype SARS-CoV-2 infection. Virus-specific T cells are detectable in most participants, albeit more variable than antibody responses. Cluster analyses of the co-evolution of antibody and T cell responses within individuals identify five distinct trajectories characterized by specific immune patterns and clinical factors. These findings demonstrate the relevant heterogeneity in humoral and cellular immunity to SARS-CoV-2 while also identifying consistent patterns where antibody and T cell responses may work in a compensatory manner to provide protection

Case Report: Stepwise Anti-Inflammatory and Anti-SARS-CoV-2 Effects Following Convalescent Plasma Therapy With Full Clinical Recovery.

In these times of COVID-19 pandemic, concern has been raised about the potential effects of SARS-CoV-2 infection on immunocompromised patients, particularly on those receiving B-cell depleting agents and having therefore a severely depressed humoral response. Convalescent plasma can be a therapeutic option for these patients. Understanding the underlying mechanisms of convalescent plasma is crucial to optimize such therapeutic approach. Here, we describe a COVID-19 patient who was deeply immunosuppressed following rituximab (anti-CD20 monoclonal antibody) and concomitant chemotherapy for chronic lymphoid leukemia. His long-term severe T and B cell lymphopenia allowed to evaluate the treatment effects of convalescent plasma. Therapeutic outcome was monitored at the clinical, biological and radiological level. Moreover, anti-SARS-CoV-2 antibody titers (IgM, IgG and IgA) and neutralizing activity were assessed over time before and after plasma transfusions, alongside to SARS-CoV-2 RNA quantification and virus isolation from the upper respiratory tract. Already after the first cycle of plasma transfusion, the patient experienced rapid improvement of pneumonia, inflammation and blood cell counts, which may be related to the immunomodulatory properties of plasma. Subsequently, the cumulative increase in anti-SARS-CoV-2 neutralizing antibodies due to the three additional plasma transfusions was associated with progressive and finally complete viral clearance, resulting in full clinical recovery. In this case-report, administration of convalescent plasma revealed a stepwise effect with an initial and rapid anti-inflammatory activity followed by the progressive SARS-CoV-2 clearance. These data have potential implications for a more extended use of convalescent plasma and future monoclonal antibodies in the treatment of immunosuppressed COVID-19 patients

Controlled nucleation of thin microcrystalline layers for the recombination junction in a-Si stacked cells

In high-efficiency a-Si : H based stacked cells, at least one of the two layers that form the internal n/p junction has preferentially to be microcrystalline so as to obtain sufficient recombination at the junction [1–6]. The crucial point is the nucleation of a very thin μc-Si : H layer on an amorphous (i-layer) substrate [2, 4]. In this study, fast nucleation is induced through the treatment of the amorphous substrate by a CO2 plasma. The resulting n-layers with a high crystalline fraction were, however, found to reduce the Voc when incorporated in tandem cells. The reduction of the Voc could be restored only by a precise control of the crystalline fraction of the n-layer. As a technologically more feasible alternative, we propose a new, combined n-layer, consisting of a first amorphous layer for a high Voc, and a second microcrystalline layer, induced by CO2 treatment, for a sufficient recombination at the n/p junction. Resulting tandem cells show no Voc losses compared to two standard single cells, and an efficient recombination of the carriers at the internal junction as proved by the low series resistance (15 Ωcm2) and the high FF ( 75%) of the stacked cells