Research Center Borstel - Leibniz Lung Center

Summary Background Rifampicin-resistant tuberculosis (RR-TB) is a major threat to public health in Viet Nam, with nearly 10,000 incident cases estimated annually. It is uncertain whether these cases are driven by transmission of resistant strains or de novo resistance acquisition during treatment. Methods We undertook dense, city-wide sampling of adults newly diagnosed with pulmonary RR-TB in Ho Chi Minh City, Viet Nam’s largest city, between March 2020 and April 2024. Participants provided sputum for culture and whole-genome sequencing (WGS), and demographic and clinical data were collected at enrolment. Phylogenetic analyses were combined with clinical histories to infer transmitted versus acquired rifampicin resistance. Estimates were corrected for sampling coverage using simulation-extrapolation (SIMEX). Temporal emergence of rifampicin resistance was reconstructed by lineage using Bayesian phylogenetic dating, and the geographic and demographic structure of transmission networks was assessed using geocoded residential data and commute time-based analyses. Findings Among 2,319 RR-TB cases diagnosed during the study period, 1,491 (64%) isolates were successfully sequenced. After accounting for sampling and phylogenetic uncertainty, we estimated that between 72% and 87% of all RR-TB arose through transmission of already-resistant strains with the remainder due to de novo acquired resistance. Bayesian dating analyses revealed that resistance emergence events occurred repeatedly from the 1980s to the present, with early events seeding long-lived, city-wide transmission networks. Transmission networks were geographically dispersed across the city, with limited household clustering, and only weakly structured by host demographics, consistent with diffuse, city-wide transmission rather than localised or assortative spread. Interpretation RR-TB in Ho Chi Minh City is driven predominantly by ongoing transmission, but a substantial minority of cases arise from newly acquired resistance. Alongside promoting early diagnosis and treatment to interrupt transmission, the main drivers of acquired resistance need to be identified to control RR-TB. Funding The Rhodes Trust, Wellcome. Research in context Evidence before this study We searched PubMed from database inception up to November 2025 for studies describing the transmission and acquisition of multidrug /rifampicin-resistant tuberculosis (MDR/RR-TB), using terms including “tuberculosis”, “rifampicin resistance”, “multidrug resistance”, “genomic epidemiology”, “whole-genome sequencing”, “transmission” and “acquired resistance”. We additionally added terms “Ho Chi Minh City” and “Viet Nam” to identify results specific our study setting. Previous studies from high burden settings typically reported that MDR/RR-TB isolates clustered with high proportions, suggesting transmission is the major contributor to MDR/RR-TB burden. However, estimates varied widely across settings, study designs and analytic approaches. Many studies relied on SNP-based clustering thresholds and did not account for incomplete sampling. Few studies integrated treatment history and homoplasy-aware phylogenetic inference or accounted for uncertainty in their estimates of the population-level contribution of de novo rifampicin resistance acquisition. No studies from Viet Nam addressed this question and the extent to which transmitted and acquired resistance contribute to the burden of MDR/RR-TB remains unknown. Added value of this study We combined dense, city-wide sampling of RR-TB in a high-burden metropolitan setting with whole-genome sequencing, historic genomic context and phylogenetic analyses to quantify the contributions of transmission and de novo acquisition to MDR/RR-TB burden. By integrating phylogenetic placement of resistance mutations with individual treatment history, we provide robust population-level estimates of transmission and independent resistance emergence while explicitly accounting for phylogenetic and sampling uncertainty. Acknowledging the potential effect of sampling coverage on these estimates, we further provide estimates extrapolated to complete coverage using a simulation-extrapolation framework. We show that the majority of RR-TB in Ho Chi Minh City is attributable to transmission, but that a substantial minority of cases reflect de novo acquisition of rifampicin resistance. Our findings demonstrate that de novo resistance acquisition continues to occur notwithstanding improvements in programmatic TB control, treatment success rates, and diagnostic capacity. Implications of all of the available evidence Our findings emphasise that MDR/RR-TB in Ho Chi Minh City is overwhelmingly driven by transmission and that controlling it requires a transmission-focused response. Intensified efforts to interrupt transmission through earlier case detection and rapid treatment initiation should be programmatic priorities. Concurrently, further investigation into the precise drivers of acquired drug resistance, which represents a potentially preventable fraction of the RR-TB burden, remains warranted

Cassie-Baxter superhydrophobic surfaces repel aqueous media by trapping a thin air layer known as plastron. However, current methods to estimate plastron characteristics on such surfaces, either directly through digital still imaging/confocal microscopy or indirectly through contact angle goniometry, are technically challenging and often imprecise. Previously, we demonstrated that the solid-liquid area fraction, which quantifies the fraction of the solid surface that is in direct contact with the surrounding liquid, can be accurately measured by reflectance optical microscopy, provided that the material surface has approximately uniform local reflectivity. To eliminate the complexity associated with optical reflectance measurements, we recently demonstrated that the drop adhesion force correlates linearly with the solid-liquid area fraction. In this study, we advance the drop adhesion force measurements obtained at different environments, compression distances, and during pressurization-hold experiments to reveal whether the plastron sustains external pressure or undergoes gradual collapse. Here, we demonstrate that measuring drop adhesion force is a robust, rapid, and technically simple method for assessing plastron properties under a wide range of environmental and surface conditions. The proposed method provides information on plastron stability within minutes and thus could aid in the design of ultrarepellent surfaces with long-term stability in demanding applications.

Kupffer cells (KCs), the prenatally seeded macrophages of the liver, display unique functional and immunophenotypic features among tissue-resident macrophages. They are considered terminally differentiated with negligible postnatal cellular input. Their adaptability in disease has thus been attributed to recruited, monocyte-derived KCs. Here, we explored KC plasticity and the impact of their ontogeny in persisting mycobacterial infections, which primarily target macrophages. Models of systemic chronic infection with Mycobacterium bovis or Mycobacterium avium were combined with genetic monocyte deficiency (Ccr2-/-, Irf8-/-), KC fate-mapping mice, bone marrow transplantation, high-resolution imaging and immunophenotyping. In addition, ATAC, bulk RNA, and single-cell RNA sequencing were conducted to specifically delineate origin, heterogeneity, and adaptations of KCs during infections. Mycobacterial infections induced the emergence of a unique KC subset, which lacked the signature markers CLEC4F and VSIG4 ("KClow"). KClow were derived from embryonically seeded KCs and accumulated exclusively within hepatic granuloma cores. In contrast, monocyte-derived macrophages were localized at the granuloma periphery, yet contributed to the tissue reaction. We identified KClow as specialized antimycobacterial effector cells with a heightened ability to produce iNOS, adapted to a hypoxic microenvironment, and able to modulate adaptive immune responses. Despite their fundamental deviation from the classical KC phenotype, KClow showed remarkable plasticity. Monocytes, on the other hand, were crucial for granuloma initiation. Mycobacterial infections reveal KCs as highly plastic cells that are critical for responding to extreme environmental changes. Kupffer cells (KCs) are traditionally regarded as terminally differentiated macrophages with limited plasticity, reliant on monocyte-derived KCs during liver infections or injury. In contrast, using a chronic mycobacterial infection model, we found tissue-resident KCs to be very plastic cells, which are strongly engaged in the antimycobacterial response and adopt a specific spatial distribution. These findings suggest that KCs respond with a unique and organ-specific program to chronic mycobacterial infections.

Rationale:Tuberculosis vaccines aim to elicit protective immunity, with IFNγ-producing CD4⁺ T cells considered central mediators of host defense. In murine models, IFNγ activates macrophages to induce inducible nitric oxide synthase (iNOS), resulting in nitric oxide–dependent control of Mycobacterium tuberculosis (Mtb).Objective:To define the relevance of IFNγ-induced iNOS activity in humans and other natural host of virulent mycobacterial species.Methods:We systematically compared IFNγ-induced effector responses in Mtb-infected myeloid cells across species. Using bulk RNA sequencing, functional infection assays, and nitric oxide measurements, we assessed IFNγ responsiveness in human and mouse macrophages. These analyses were extended to monocytes from seven mammalian species and complemented by reanalysis of publicly available single-cell RNA-sequencing datasets and spatial proteomic imaging of tuberculous granulomas.Measurements and Main Results:Single-cell transcriptomic reanalysis revealed minimal NOS2 expression in myeloid cells from human and non-human primate granulomas. In vitro, IFNγ pretreatment failed to induce NOS2 transcription, iNOS activity, or Mtb growth restriction in human macrophages, in stark contrast to murine cells. Across species, iNOS activity was largely restricted to mice, with limited induction in cattle monocytes. Instead, human respiratory epithelial cells consistently expressed NOS2, and multiplexed ion beam imaging localized iNOS protein to epithelial compartments adjacent to granulomatous lesions.Conclusion:IFNγ signaling is uncoupled from iNOS induction in primate myeloid cells and epithelial compartments represent dominant sources of iNOS in human tuberculosis. These findings, challenge murine macrophage–centric paradigms and IFNγ-based correlates used in TB vaccine development and central to pathophysiology of tuberculosis and other pneumonias.

The methylerythritol phosphate (MEP) pathway is essential for isoprenoid biosynthesis in many pathogenic bacteria but is absent in humans, making its enzymes attractive antibacterial targets. IspE catalyzes the ATP-dependent phosphorylation of 4-diphosphocytidyl-2-C-methylerythritol, a key step in this pathway. Using a previously identified optimized hit as a starting point, we designed and synthesized a focused library of twelve simplified analogues that retained essential pharmacophoric features while improving synthetic accessibility. Docking studies with Escherichia coli IspE guided the design and predicted binding orientations consistent with known ligand interactions. Biochemical evaluation of the library against E. coli and Klebsiella pneumoniae IspE revealed several low-micromolar inhibitors, confirming the predicted binding interactions. Structure–activity relationships indicated that the hydrophobic pocket adjacent to the cytidine-binding region is a key determinant of potency. Although the compounds showed limited whole-cell activity, these results demonstrate that simplified amide analogues can effectively engage the IspE active site and highlight the importance of the hydrophobic pocket in ligand binding. Overall, this work combines structure-based design, synthesis, and biochemical validation to provide a foundation for further optimization of simplified IspE inhibitors as potential antibacterial leads.

Abstract Background Mycobacterium tuberculosis (Mtb) causes significant morbidity and mortality with worse outcomes in individuals infected with multi-drug resistant Mtb (MDR-TB). Specific MDR-TB strains have shown additional virulence factors that could prompt altered host immune response in comparison to drug susceptible Mtb (DS-TB). We evaluated the immune response of individuals with MDR-TB in Callao, Peru compared to DS-TB.Mtb-specific IL-17 production from PBMC collected from participants newly diagnosed with MDR-TB and DS-TB.Boxes show mean, min, and max values of fold change in IL-17 between ESAT-6·CFP-10-stimulated vs. unstimulated conditions. *p < 0.05 Methods Adults with a new diagnosis of MDR-TB and DS-TB were enrolled from tuberculosis clinics in Callao, Peru while healthy control participants were recruited from local medicine clinics and community. Participants provided blood samples for peripheral blood mononuclear cells (PBMCs) and plasma isolation. Soluble immune markers including proinflammatory cytokines and immunoglobulins were tested in plasma. PBMCs were analyzed via flow cytometry for immunophenotyping and via cell stimulation assays. To account for false positive rates with 75 distinct cell populations in the immunophenotyping assay Benjamini-Hochberg correction was employed. Results Analysis of cell phenotypes identified notable differences. CD8+ T cells were increased in patients with MDR-TB compared to DS-TB (medians (interquartile range) [n]: 51.9 (31.2-65.7) [14] and 27.8 (23.2-34.8) [22], p = 0.036)), and the frequency of CD8+/CD38+ cells were decreased in MDR-TB compared to DS-TB (4.47 (3.3-6.6) [14] and 9.4 (6.2-13.9) [22], p = 0.0057). While analysis of soluble plasma factors did not show significant differences, analysis of cell stimulation assays showed an increased IL-17 production in PBMC from participants with MDR-TB compared to DS-TB when stimulated with the Mtb antigen ESAT6/CFP10 (see Figure; p=0.015). Conclusion While much of the immune response to MDR-TB appears similar to DS-TB, we found notable differences, with increased CD8+ T cells, decreased CD8/CD38+ T cells, and increased IL-17 production in response to Mtb stimulation. These data suggest that MDR-TB could prompt differential immune signatures, including expanded IL-17 responses. Further work may lead to better understanding of host-pathogen interactions in MDR-TB disease and possible optimized therapies. Disclosures All Authors: No reported disclosures

Solid organ transplant (SOT) recipients face elevated tuberculosis risk, yet optimal prevention strategies in low- to medium-incidence regions remain unclear. We conducted a multicenter retrospective cohort study of adult SOT recipients transplanted between 2007 and2012 at 15 European centers, with follow-up through 2018. The primary outcome was microbiologically confirmed post-transplant tuberculosis. Incidence rates were calculated per 100,000 person-years; standardized incidence ratios (SIRs) used World Health Organization country-specific background rates. Cox models assessed risk factors. Among 5805 patients (median age 51; 62.7% male; 73.9% renal transplants), 33.8% were tested for tuberculosis infection and 10.3% received tuberculosis preventive therapy (TPT). Over 33,785 person-years, 23 patients (0.4%) developed tuberculosis (68.0/100,000 person-years). Highest incidence occurred in patients with positive screening but no TPT (233.8/100,000). Incidence was higher in Southern vs. Central Europe (251.9 vs. 28.7/100,000), with pooled SIRs of 12.8 and 3.1, respectively. Tuberculosis risk was elevated among Southern European recipients (HR 22.9) and those with migration history (HR 2.7). Tuberculosis risk is increased in European SOT recipients. Regionally adapted prevention strategies, including targeted screening in low-incidence areas and universal screening in higher-incidence regions, are warranted.

PfEMP1 is a variable antigen displayed on erythrocytes infected with the malaria parasite Plasmodium falciparum. PfEMP1 mediates binding of the infected cell to the endothelium of blood vessels, a cause of severe malaria. Each parasite encodes ~60 different PfEMP1 variants but only one is expressed at a time. Switching between variants underlies immune evasion in the host and variant-specific severity of disease. PfEMP1 is difficult to study due to expression heterogeneity between parasites which also renders genetic modification approaches ineffective. Here, we used selection-linked integration (SLI) to generate parasites all expressing the same PfEMP1 variant and genome edit the expressed locus. Moving this system from the reference strain 3D7 to IT4 resulted in PfEMP1 expressor parasites with effective receptor binding capacities. We also introduce a second version of SLI (SLI2) to introduce additional genome edits. Using these systems, we study PfEMP1 trafficking, generate cell lines binding to the most common endothelial receptors, survey the protein environment from functional PfEMP1 in the host cell, and identify new proteins needed for PfEMP1-mediated sequestration. These findings show the usefulness of the system to study the key virulence factor of malaria parasites.

Multiplex immunofluorescence and immunohistochemistry (mIF/IHC) are increasingly employed antibody-based technologies that use tissue sparingly and facilitate the detection of co-localized or neighboring biomarkers. Specifically, these platforms enable spatial analyses of the tumor microenvironment as well as extended applications, for example, describing normal tissue anatomy, autoimmunity, infectious diseases, etc. mIF/IHC has greatly enhanced biomarker discovery efforts, and a growing number of studies suggest superiority to traditional IHC. Standardization of staining approaches, reporting of image analysis strategies and resultant data is critical for facilitating cross-study comparisons, validation, deployment, and generalization of findings. To address this challenge, The Society for Immunotherapy of Cancer (SITC) previously published two articles providing best practice guidelines for mIF/IHC staining, image analysis, and data sharing. Here, SITC convened stakeholders to develop the third article in the series, a consensus checklist for scientific reporting of mIF/IHC data to support and complement the best practice guidelines. The checklist includes critical components of mIF/IHC applications to be defined within publications such as detailed descriptions of analytical validation; image acquisition, selection, and registration methods; and cell clustering and spatial analysis strategies, amongst others. Such information will help with data reproducibility and comparison across studies towards future drug and assay development.