Citations

Abstract Introduction Screening individuals with latent tuberculosis infection (LTBI) from regions with varying TB endemicity is critical for global TB control. Immune responses to TB differ due to variations in exposure, immune adaptations, and environmental factors. This study evaluated antigen-specific immune responses in untreated LTBI individuals from low- and high-TB-endemic settings. Methods Peripheral blood mononuclear cells from untreated LTBI individuals from low-endemic (USA) and high-TB-endemic settings (Mexico) were stimulated with PPD, MTB300, ESAT-6/CFP-10 (RD1) peptides, mycobacterial glycopeptidolipids (GPL), and controls. We compare flow cytometric immune responses of T-cell intracellular cytokines and activation markers using non-parametric statistical methods. Results We studied age-matched, untreated LTBI individuals who tested IGRA(+)/TST(+) from low-TB-endemic (n = 18) and 18 healthcare workers from a high-TB-endemic settings. IGRA results showed no statistical differences across groups. The frequency of CD3+CD8+IFN-γ+HLA-DR+ and total CD3+TNF-α+ T cells after RD1 stimulation was significantly higher in the high-TB-endemic group (P < 0.05). Additionally, we observed significant increased frequencies in CD3/CD4+TNF-α+HLA-DR+ and CD8+TNF-α+HLA-DR+ T cells stimulated by GPL in the same group, probably associated with nontuberculous mycobacteria (NTM) exposures. Our findings suggest possible recent TB and NTM exposures in healthcare workers from a high-TB-endemic setting, resulted in increased antigen-specific adaptive TNF-α and IFN-γ producing T cells, which are important for pathogen control and immunity. Conclusion We found significant differences in antigen-specific cell immunity, including HLA-DR activated T-cells, a marker of recent antigen encounter, in individuals from a high-TB-endemic settings. These findings underscore the importance of settings-specific immune biomarkers for improving LTBI diagnostics, clinical management, and vaccination trials. Funding Source National Institute of Allergy and Infectious Diseases at the National Institutes of Health Topic Categories Immune Mechanisms of Human Disease (HUM)

Abstract Rationale Blood eosinophil count (BEC) has emerged as a marker of inflammatory endotypes in bronchiectasis. We examined whether bacterial and mold respiratory culture profiles differ by BEC, comparing patients with BEC ≥300 cells/μL to those with lower BEC. Methods We analyzed patients with bronchiectasis evaluated at a specialized clinic (2019-2025) who had contemporaneous respiratory bacterial and fungal cultures and BEC. Patients were stratified by BEC <300 vs ≥ 300 cells/μL; those with BEC <300 were further subclassified (<100 and 100-299 cells/μL) for figure presentation only. Cultures were evaluated for overall growth and organism-specific prevalence across eosinophil strata, focusing on the most frequently isolated organisms. Group comparisons were performed using Fisher’s exact test. Results A total of 608 patients met inclusion criteria (median age 69 years [IQR 61-77], 72% female); 138 (22.7%) had BEC ≥300 cells/μL. Bacterial growth occurred in 277 patients (45.6%) and mold growth in 366 (60.2%). The odds of bacterial growth were similar between BEC ≥300 and <300 groups (47.1% vs 45.2%). In contrast, BEC ≥300 was associated with higher odds of mold growth (68.1% vs 58.0%; OR 1.54, p = 0.038). Pseudomonas species (18.9%) were the most common bacterial isolates and comprised a greater proportion of all bacterial isolates in patients with BEC ≥300 compared with lower BEC (44.7% vs 30.0%; OR 1.89, p = 0.019). Penicillium species (27.5%), followed by Aspergillus (26.9%), were the most frequently isolated molds, with no differences in distribution across BEC strata. Conclusions BEC ≥300 cells/μL was associated with increased odds of respiratory mold growth and enrichment of Pseudomonas species, suggesting that eosinophil-defined endotypes may identify patients with distinct airway microbiologic patterns that could inform future phenotyping and treatment strategies. This abstract is funded by: None

Purpose Mycobacterium avium complex lung disease (MAC-LD) represents the most common form of nontuberculous mycobacterial lung infection, particularly affecting women and older adults. Clinical management remains challenging, including prediction and differentiation between progressive and non-progressive disease. Overexpression of PD-1 pathways have been reported in progressive disease, and we hypothesize that PD-1 inhibition can restore antigen-specific immune responses detectable by FluoroSpot assay. Methods Peripheral blood mononuclear cells from 22 participants, including progressive and non-progressive MAC-LD patients, healthy controls, and individuals with latent tuberculosis infection (LTBI) were stimulated with nontuberculous mycobacteria and Mycobacterium tuberculosis antigens. Cytokine secretion of interferon-gamma (IFN-γ) and interleukin-2 (IL-2) were quantified using dual FluoroSpot assay, with and without PD-1 blockade. Results Progressive MAC-LD cases demonstrated significant increases in glycopeptidolipid (GPL)- and PPD-stimulated IFN-γ responses following PD-1 blockade, which was not seen in healthy controls, LTBI, or non-progressive MAC-LD patients. After GPL stimulation and in the presence of anti-PD-1, the number of IFN-γ responses increased from 25% to 75% in samples from progressive MAC-LD patients. This supports an association between PD-1–mediated T-cell dysfunction and disease progression. Mycobacterial MTB300 peptide pool antigen failed to discriminate MAC-LD from healthy controls, underscoring antigen specificity. Conclusions Our pilot study demonstrates that IFN-γ FluoroSpot assays, particularly when combining GPL or PPD stimulation and PD-1 inhibition, may help identify progressive MAC-LD. These findings also support other studies reporting that immune checkpoint dysregulation can be present in some patients with progressive and severe MAC-LD patients. Additional studies are warranted to determine its pathogenic role.

Abstract Background Sputum color is a visual marker of airway inflammation and disease activity in bronchiectasis, ranging from clear or light gray (mucoid) to yellow-green (purulent), the latter largely reflecting neutrophil myeloperoxidase release. This study evaluated associations between sputum color and common clinical markers of disease activity to determine which parameters differed most between color categories. Methods Forty spontaneous sputum samples were analyzed from 80 consecutive adults with non-cystic fibrosis bronchiectasis (median age 70 years [IQR 12]; 74% female) enrolled between January 2024 and April 2025 in an observational study. Sputum color was graded using the Murray Sputum Color Chart and classified as mucoid (color #1) or purulent (colors #2-4). Clinical parameters, including blood cell counts, C-reactive protein (CRP), fractional exhaled nitric oxide (FeNO), and results of bacterial and acid-fast bacillus cultures (induced or bronchoscopic), were obtained from medical records if available within one month of enrollment. Categorical variables were analyzed using chi-square or Fisher’s exact tests and continuous variables with the Mann-Whitney U test. A secondary analysis compared patients who could versus could not produce spontaneous sputum. Results Of 40 samples, 20 (50%) were purulent. Blood neutrophil counts (median 4.5 vs. 3.9 x109/L), neutrophil-to-lymphocyte ratios (2.5 vs. 2.6), and eosinophil counts (1.7 vs. 1.7 x109/L) did not differ significantly between groups (n = 26). FeNO was normal in all 10 patients with available data and was not analyzed further. CRP was higher among patients with purulent sputum (4.6 vs. <3 mg/L; n = 20; p = 0.044). Bacterial growth was more frequent in purulent than mucoid samples (73% vs. 31%; n = 31; p = 0.032). Isolates in the purulent group included Klebsiella, Pseudomonas, Staphylococcus, and Streptococcus (n = 2 each), and single isolates of Acetobacter, Acinetobacter, Chryseobacterium, Delftia, Enterobacter, Escherichia, and Moraxella. In the mucoid group, Pseudomonas, Staphylococcus (n = 2 each), and Stenotrophomonas (n = 1) were identified. Mycobacterial growth did not differ significantly (20% vs. 12.5%; n = 31; p = 0.65). Patients unable to produce spontaneous sputum had a higher rate of mycobacterial isolation on induced/bronchoscopic samples (43% vs. 16%; n = 54; p = 0.027), though other parameters were similar. Over 90% of measurements were obtained within three days of enrollment, and freezing did not appear to alter sputum color. Conclusions Even within a small cohort, purulent sputum was associated with higher CRP and greater bacterial growth, supporting sputum color as a rapid, inexpensive marker of disease activity in bronchiectasis. Further research should clarify whether sputum color primarily reflects bacterial colonization, airway inflammation, or both. This abstract is funded by: None

Introduction Asymptomatic individuals with negative Interferon-γ release assay (IGRA) and tuberculin skin test (TST) results are often labeled as tuberculosis-unexposed individuals. We hypothesize that flow cytometric (FC) detection of antigen-stimulated T-cells in peripheral blood can differentiate IGRA(-)/TST(-) individuals with and without prior potential M. tuberculosis (Mtb) exposure. Methods We used FC multiparameter profiling of T-cell activation markers in TST/IGRA-negative individuals at risk for Mtb exposure and in unexposed controls. Results We studied 54 IGRA(-)/TST(-) subjects, including 27 individuals at risk for prior Mtb exposure. While quantitative IGRA results were negative in all patients, FC profiling of CD3/CD4 + IFN-γ + HLA-DR + T-cells after stimulation with a pool of Mtb-specific region of difference-1 (RD1) peptides, mycobacterial (PPD and MTB300), or Candida antigens, statistically differentiated between unexposed and potentially exposed individuals (P<0.05). ROC analysis showed PPD and RD1 peptides responses had AUCs >0.7 (sensitivity of 55.5–74% and specificity of 66.6–85.1%). The presence of a polyfunctional RD1-reactive CD3/CD8 + IFN-γ + TNF-α + T-cell subset also significantly differentiated these groups (AUC 0.66). Other T-cell markers demonstrated no significant differences. Conclusions FC detection of antigen-specific IFN-γ + HLA-DR + CD4+T-cells and IFN-γ + TNF-α + CD8 + T-cells can differentiate IGRA(-)/TST(-) individuals at risk of Mtb exposure from unexposed controls. Further validation is needed to determine whether this immunoprofiling approach can improve detection of Mtb infections and identification of unexposed individuals for future biomarker studies and vaccine trails.

Abstract Introduction/Objective Cervical cancer remains a global health concern, with significantly higher incidence in countries like Peru compared to the U.S. Cytopathology plays a vital role in early detection and prevention, making it an important area of focus for aspiring pathologists. Methods/Case Report A medical student with an interest in pathology participated in a global health rotation in Cusco, Peru, at CerviCusco, a nonprofit clinic dedicated to cervical cancer care. Under the guidance of a trained cytotechnologist, the student engaged in hands-on cytopathology work, including processing and screening SurePath (Franklin Lakes, NJ) slides, and reviewing abnormal cases. The student had access to a microscope and dedicated time developing microscopy skills and learning cervical cytomorphology, with emphasis on identifying significant abnormalities. Results NA Conclusion This experience demonstrates the educational value of incorporating global health electives into medical training, especially in resource-limited settings with high disease burden. Exposure to such environments enhances foundational diagnostic skills and fosters an early appreciation for global health disparities. Expanding similar opportunities can enrich pathology education and inspire future contributions to international health efforts.

Importance Advances in Alzheimer disease (AD) have shifted research focus to earlier disease stages, necessitating more scalable approaches to identify cognitively unimpaired individuals with amyloid β (Aβ) pathology. Objective To assess the utility of plasma phosphorylated tau 217 (p-tau217) for classifying Aβ status in cognitively unimpaired individuals, both as a stand-alone test and in a 2-step approach where positive plasma results were confirmed using a second modality (Aβ positron emission tomography [PET] or cerebrospinal fluid [CSF]). Design, Setting, and Participants This cross-sectional cohort study used data collected between June 2009 and March 2024. We included 2916 cognitively unimpaired participants from 12 international independent observational cohorts in the US, Europe, Australia, and Canada with available plasma p-tau217 levels and CSF or PET Aβ biomarkers. Performance comparisons between mass spectrometry and immunoassay-based p-tau217 measurements were also performed (n = 964). Exposures Plasma p-tau217 levels measured by immunoassay. Main Outcome and Measures Aβ status, determined by CSF or Aβ PET biomarkers. Results Participants had a mean (SD) age of 66.9 (9.9) years; 971 (33.3%) were Aβ positive by either CSF or PET, 1667 (57.2%) were women, and 1108 (38.1%) carried at least 1 APOE ε4 allele. As a stand-alone test, plasma p-tau217 achieved a positive predictive value (PPV) of 79% (95% CI, 74-84) and an overall accuracy of 81% (95% CI, 80-82). In a 2-step workflow, the PPV and accuracy significantly increased to 91% (95% CI, 86-95). While this approach required screening of 677 individuals with plasma p-tau217 to identify 100 Aβ-positive individuals, compared to 536 participants when using PET alone, it reduced the need for PET testing to 124. Immunoassays demonstrated comparable PPVs to mass spectrometry (80% [95% CI, 74-86] vs 85% [95% CI, 81-90]; P = .12) but significantly lower overall accuracy (82% [95% CI, 79-84]% vs 88 [95% CI, 86-90]; P < .001) and true Aβ-positive detection rate (49% [95% CI, 43-55] vs 69% [95% CI, 64-75]; P < .001). Conclusions and Relevance The findings highlight the potential of plasma p-tau217 as a stand-alone test—or when used in a sequential 2-step approach alongside PET or CSF testing—as a cost-effective, scalable, and minimally burdensome strategy for identifying preclinical AD. Tailored screening workflows that incorporate p-tau217 can improve efficiency in participant selection for preclinical AD trials and, in the future, help guide access to disease-modifying treatments.