Commercial availability of serological tests to evaluate immunoglobulins (Ig) targeting severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has grown exponentially since the start of the coronavirus disease 2019 (COVID-19) outbreak. Thorough validation of these tests is important before use as epidemiological tools to infer seroprevalence in specific populations and as diagnostic tools to complement molecular approaches (e.g., quantitative reverse transcription-polymerase chain reaction). Commercial serological tests from 11 suppliers were assayed side-by-side using 126 samples from SARS-CoV-2-infected inpatients and 36 from healthy and HIV-infected individuals. The majority of the tests assayed have >95% specificity. For the sensitivity calculation, samples were stratified by days since symptoms onset; sensitivity peaks at 16–21 days for IgM and IgA (maximum 91.2%, Euroimmun) and, dependant on the test, at 16–21 or >21 days for IgG (maximum 94.1%, Snibe). Data from semiquantitative tests show that patients with a severe clinical presentation have lower levels of Ig targeting SARS-CoV-2 at 21 days, compared to patients with a non-severe presentation. This study highlights the heterogeneity of sensitivity and generally high specificity of the serological tests and establishes a basis for their usefulness to complement diagnostic techniques and population seroprevalence studies.

The importance of circulating immune cells to primary progressive multiple sclerosis (PPMS) pathophysiology is still controversial because most immunotherapies were shown to be ineffective in treating people with PPMS (pwPPMS). Yet, although controversial, data exist describing peripheral immune system alterations in pwPPMS. This study aims to investigate which alterations might be present in pwPPMS free of disease-modifying drugs (DMD) in comparison to age- and sex-matched healthy controls. A multicentric cross-sectional study was performed using 23 pwPPMS and 23 healthy controls. The phenotype of conventional CD4⁺ and CD8⁺ T cells, regulatory T cells (Tregs), B cells, natural killer (NK) T cells and NK cells was assessed. Lower numbers of central memory CD4⁺ and CD8⁺ T cells and activated HLA-DR⁺ Tregs were observed in pwPPMS. Regarding NK and NKT cells, pwPPMS presented higher percentages of CD56^dimCD57⁺ NK cells expressing NKp46 and of NKT cells expressing KIR2DL2/3 and NKp30. Higher disease severity scores and an increasing time since diagnosis was correlated with lower numbers of inhibitory NK cells subsets. Our findings contribute to reinforcing the hypotheses that alterations in peripheral immune cells are present in pwPPMS and that changes in NK cell populations are the strongest correlate of disease severity.

Commercial availability of serological tests to evaluate immunoglobulins (Ig) towards severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has grown exponentially since the onset of COVID-19 (Coronavirus Disease 2019) outbreak. Their thorough validation is of extreme importance before using them as epidemiological tools to infer population seroprevalence, and as complementary diagnostic tools to molecular approaches (e.g . RT-qPCR). Here we assayed commercial serological tests (semiquantitative and qualitative) from 11 suppliers in 126 samples collected from hospitalized COVID-19 patients, and from 36 healthy and HIV-infected individuals (collected at the pre-COVID-19 pandemic). Specificity was above 95% in 9 tests. Samples from COVID-19 patients were stratified by days since symptoms onset (21 days). Tests sensitivity increases with time since symptoms onset, and peaks at 16-21 days for IgM and IgA (maximum: 91.2%); and from 16-21 to >21 days for IgG, depending on the test (maximum: 94.1%). Data from semiquantitative tests show that patients with severe clinical presentation have lower relative levels of IgM, IgA and IgG at 21 days since symptoms onset the relative levels of IgM and IgG (in one test) are significantly higher in patients with severe clinical presentation, suggesting a delay in the upsurge of Ig against SARS-CoV-2 in those patients. This study highlights the high specificity of most of the evaluated tests, and sensitivity heterogeneity. Considering the virus genetic evolution and population immune response to it, continuous monitoring of commercially available serological tests towards SARS-CoV-2 is necessary.

Tuberculosis (TB) remains a major global health challenge, with current diagnostic tools unable to reliably distinguish between individuals at different stages of the TB spectrum of infection (TBI) and disease (TBD). Interferon-gamma release assays (IGRAs), while commonly used, are limited by their reliance on a single cytokine and inability to differentiate disease states. In this study, we evaluated a multiplex FluoroSpot assay that simultaneously detects interferon gamma (IFNγ), interleukin (IL)2, and tumor necrosis factor (TNF)-secretion in response to Mycobacterium tuberculosis ( Mtb ) antigens ESAT-6, CFP-10, and EspC. PBMCs from individuals with TBD ( n = 24), TBI ( n = 64), and IGRA-negative controls ( n = 26) were analyzed. The assay demonstrated high sensitivity and specificity, particularly through the detection of triple cytokine-secreting T cells (IFNγ/IL-2/TNF), minimizing false-positive rates. Moreover, distinct cytokine secretion patterns were associated with different stages of infection. Individuals with TBD had more IFNγ/TNF-producing cells, while those with TBI had more cells producing IFNγ/IL-2 or IL-2/TNF. Separating individuals with TBI based on time since likely Mtb exposure revealed that more recent infection was associated with reduced IFNγ responses and a higher proportion of IL-2/TNF-secreting cells. The use of spot volume analysis provided semi-quantitative insights into cytokine production. These findings suggest that multiplex cytokine profiling can improve TB diagnostics and offer a deeper understanding of host immune responses across the TB spectrum. Including non-IFNγ responses may be particularly valuable in identifying individuals at higher risk of disease progression, such as recently infected individuals, and in populations where current diagnostics underperform. IMPORTANCE: Accurate diagnosis of Mycobacterium tuberculosis ( Mtb ) infection remains a cornerstone in tuberculosis (TB) control. Current interferon-gamma release assays (IGRAs) lack the ability to distinguish between individuals at different stages of the TB infection spectrum, limiting their utility. This study evaluates a multiplex FluoroSpot assay that simultaneously detects interferon gamma (IFNγ), interleukin 2, and tumor necrosis factor secretion in response to Mtb -specific antigens ESAT-6, CFP-10, and EspC. The assay demonstrated improved performance compared to standard IGRA methods, particularly through the identification of triple cytokine-secreting T cells. Importantly, it revealed distinct cytokine profiles associated with different stages of TB infection, offering potential for improved risk stratification and infection monitoring. These findings support the FluoroSpot assay as a promising tool for enhancing TB diagnostics and understanding host immune responses. Its application could be especially valuable in contact tracing, where Mtb -specific T-cell responses may not yet produce detectable amounts of IFNγ.

Objective: To assess the peripheral immune system of newly diagnosed relapsing remitting multiple sclerosis (RRMS) patients and compare it to healthy controls (HC). Methods: Cross-sectional study with 30 treatment-naïve newly diagnosed RRMS patients, and 33 sex and age-matched HC. Their peripheral blood mononuclear cells were analysed regarding: i) thymic function surrogates [T cell receptor excision circles (TRECs) and recent thymic emigrants (RTEs)]; ii) naïve and memory CD4 ⁺ and CD8 ⁺ T cells subsets; iii) T helper (Th) phenotype and chemokine receptors expression on T cells subsets; iv) regulatory T cell (Tregs) phenotype; and vi) expression of activating/inhibitory receptors by natural killer (NK) and NKT cells. Analyses were controlled for age, sex, and human cytomegalovirus (HCMV) IgG seroprevalence. Results: Newly diagnosed RRMS patients and HC have equivalent thymic function as determined by similar numbers of RTEs, and levels of sjTRECs, DJβTRECs and sj/DJβTREC ratio. In the CD8 ⁺ T cells compartment RRMS patients have a higher naïve/memory ratio and lower memory cell counts in blood, specifically of effector memory and TemRA CD8 ⁺ T cells. Among CD4 ⁺ T cells lower blood counts of effector memory cells are found in patients upon controlling for sex, age and HCMV IgG seroprevalence. RRMS patients have higher percentage of naive Tregs comparing to HC. Percentages of immature CD56 ^bright NK cells expressing the inhibitory receptor KLRG1, and of mature CD56 ^dim CD57 ⁺ NK cells expressing NKp30 are higher in patients. No major alterations are observed on NKT cells. MS severity and time from relapse correlate with immune cells alterations. Conclusion: Characterization of the peripheral immune system of treatment-naïve newly diagnosed RRMS patients unveiled immune features present at clinical onset including lower memory T cells blood counts, particularly among CD8 ⁺ T cells, higher percentage of naïve Tregs and altered percentages of NK cells subsets expressing inhibitory or activating receptors. These findings might set the basis to better understand disease pathogenesis.