Background/Objectives: Hepatitis E virus (HEV) is one of the leading causes of acute hepatitis, with immunosuppressed individuals, such as oncology patients, being particularly vulnerable to chronic infections that may progress to liver disease or fatal outcomes. Assay variability complicates HEV prevalence assessment in at-risk groups. This study aimed to compare the reliability and concordance of three HEV antibody assays—Wantai, Euroimmun, and Elecsys^®—in immunosuppressed oncology patients. Methods: In this prospective pilot study, serum samples were obtained from oncology patients between September 2020 and October 2021. Samples were collected both at baseline (treatment-naive) and during ongoing treatment. A healthy control group was retrospectively included for comparative analysis. Anti-HEV IgM and IgG antibodies were tested in all samples using enzyme-linked immunosorbent assays (Wantai, Euroimmun) and an electrochemiluminescence immunoassay (Elecsys^®). Demographic and clinical data, along with information on HEV risk factors, were extracted from medical records and patient questionnaires. Results: HEV IgM prevalence ranged from 0% (Wantai) to 6% (Elecsys^®), while IgG prevalence was 12% (Euroimmun), 38% (Wantai), and 53% (Elecsys^®). Concordance was poor, with Cohen’s Kappa values indicating slight to moderate agreement (κ = 0.000–0.553). Patients with hematological malignancies exhibited the highest IgG seroprevalence. Risk factor analysis revealed the highest association between HEV exposure and the consumption of undercooked pork or crop-based agriculture. Conclusions: Significant variability among HEV serological assays highlights the challenges of reliable HEV diagnostics in immunosuppressed oncology patients. Assay selection and improved testing strategies are critical for this high-risk group.

Background/Objectives: Pneumocystis jirovecii pneumonia (PJP) is the most frequently diagnosed AIDS-defining illness in Europe, with especially high mortality in HIV-negative patients caused by delayed diagnosis and low awareness. This study aims to evaluate cytokine release assays (CRA) to facilitate a less invasive and resource-efficient PJP specific diagnostic test. We focus on the P. jirovecii antigens Kexin 1 (KEX1), MSG1, and MSG2, which were identified in prior studies as immunologically relevant. Methods: Whole blood samples from 50 participants—22 healthy individuals and 28 immunocompromised individuals, including 8 with proven PJP—were stimulated in vitro with full-length and partial KEX1, MSG1, MSG2, and a combination of all three antigens (PJ-MIX). Following 24 h incubation at 37 °C, cytokine levels of IL-2, IFN-γ, IL-17A, and IL-17F were measured. Results: Stimulation with full-length KEX1, MSG1, MSG2, and PJ-MIX antigens induced higher IL-2 concentrations in the healthy control group compared to the groups IL-2 baseline levels and to the group of proven PJP cases. Similarly, stimulation with full-length KEX1, MSG1, and PJ-MIX elevated IFN-γ levels in the healthy control group compared to baseline IFN-γ levels. Conclusions: Our findings highlight the potential of IL-2 and IFN-γ release following stimulation with PJ antigens, with PJ-MIX eliciting the strongest and most significant responses, suggesting a cumulative antigen effect. This pilot study establishes a foundation for a PJP-specific CRA, deepening our knowledge of T-cell immunity against PJP. Clinically, such a test could, among other applications, evaluate at-risk patients who should receive prophylaxis and may consequently reduce PJP-related morbidity and mortality.

Background: Improving precision medicine in chemotherapy requires highly sensitive and easily applicable diagnostic tools. In addition, non-invasive molecular real-time monitoring of cytotoxic response is highly desirable. Here, we employed the kinetics of DNA double-strand breaks (DSB) and cell-free DNA (cfDNA) in a cell model of topoisomerase II-inhibitors in T cell leukemia (Jurkat cells) compared to normal cells (peripheral blood mononuclear cells, PBMCs). Methods: We applied automated microscopy to quantify immuno-stained phosphorylated H2AX (γH2AX) as a marker for either DNA damage response (DDR) or cell death and quantitative PCR-based analysis of nuclear and mitochondrial cfDNA concentrations. Results: Jurkat cells displayed a DDR to cytotoxic drug treatment significantly earlier than PBMCs, and etoposide (ETP) induced DSB formation faster than doxorubicin (DOX) in both Jurkat and PBMCs. Jurkat cells exhibited an earlier cytotoxic response compared to PBMC, with a significantly increased mitochondrial cfDNA formation after 2 h of DOX application. In PBMCs, increased cell death was detected after 4 h of incubation with ETP, whereas DOX treatment was less effective. Conclusions: Both automated microscopy and mitochondrial cfDNA quantification analysis indicate that (malignant) Jurkat cells are more sensitive to DOX than (healthy) PBMC. Our real-time approach can improve DDR inducing drug selection and adaptation in cancer therapy and aids in decisions for optimal patient biosampling.