Background: Drug-resistant strains of Leishmania infantum challenge the effectiveness of treatments for clinical leishmaniosis and may lead to more frequent relapses. Copy number variation (CNV) at specific genetic loci is associated with drug resistance and virulence, but information about its prevalence in endemic regions is limited. This study examines the drug resistance and virulence status of Leishmania strains in human and canine isolates from the Mediterranean region. Methods: Forty-eight Leishmania infantum isolates were whole-genome sequenced with nanopore long reads, followed by de novo assembly. We analyzed chromosomal aneuploidies and gene copy number variation in loci linked to drug resistance and virulence in Leishmania , alongside the genomic structure and rearrangements responsible for these variations. Results: Complete genomes were de novo assembled for 35 L. infantum isolates (22 from dogs and 13 from humans), revealing significant chromosomal variability. We assessed copy number variation for 22 potential biomarkers: 15 genes related to drug resistance to first-line drugs (METK for allopurinol; LdSMT for amphotericin B; AQP1 and H-locus for antimonials; LdMT, LdRos3, and MSL for miltefosine; and PPM for paramomycin) and 7 genes related to virulence (lipophosphoglycan and proteophosphoglycan biosynthesis, and the Lack protein). Drug-resistance biomarkers were identified in 80% of the isolates. Canine strains primarily showed resistance to allopurinol and antimonials, while human isolates exhibited a broader resistance spectrum, especially to antimonials and paromomycin. The co-occurrence of resistance biomarkers was common, especially for allopurinol and antimonial resistance. Distinct mechanisms underlie the observed copy number variations. Virulence-associated genes were less variable among isolates. Conclusions: The prevalence of drug-resistance biomarkers in Leishmania infantum strains from the Mediterranean region, as revealed by this study, underscores the critical need for routine resistance surveillance in managing clinical leishmaniosis. These findings not only inform current clinical practice but also pave the way for more effective management strategies in the future. Graphical

Tricyclic and tetracyclic lactone derivatives of thieno[2,3-b]pyrazine or thieno[2,3-b]quinoline, and 2H-pyrones were prepared using different methodologies. Pd/Cu-catalyzed Sonogashira coupling using Et₃N as a base, of methyl 7-bromothieno[2,3-b]pyrazine-6-carboxylate and (het)arylalkynes to yield the Sonogashira ester products, gave also the corresponding tricyclic lactones as minor products. However, the major products did not cyclize with TFA. Tricyclic lactones were then obtained by a tandem one-pot Sonogashira coupling and 6-endo-dig lactonization of 7-bromothieno[2,3-b]pyrazine-6-carboxylic acid with (het)arylalkynes, in good yields. Halogenated tricyclic lactones were synthesized by halocyclization using CuX and NXS. Tetracyclic lactones were synthesized through a Rh(III)-catalyzed formal [4+2] cycloaddition, between thieno[2,3-b]quinoline-2-carboxylic acid and internal alkynes, triggered by C-H activation, with the carboxylic group acting as a directing group. Using the SRB assay, the antitumor activity of both Sonogashira products and lactones was evaluated across five human cancer cell lines (CaCo-2, MCF-7, AGS, HeLa, NCI-H460). The best-performing compound was a Sonogashira product showing a GI₅₀ < 10 µM in all tumor cell lines and low toxicity in PLP2 cells. Additionally, antiparasitic testing against Trypanosoma brucei and Leishmania infantum revealed some compounds with IC₅₀ < 11 µM, though some level of cytotoxicity was observed in THP-1—derived macrophages.

Leishmania is an intracellular protozoan parasite that causes leishmaniasis, a disease prevalent in 97 countries. Co-infection with HIV increases susceptibility to visceral leishmaniasis (VL), accelerating HIV’s progression to AIDS. Managing VL in HIV-infected individuals is challenging due to atypical presentations and limited therapeutic responses, highlighting the need to develop new disease management strategies. Extracellular vesicles (EVs) hold great promise for this goal as they can be used for a higher understanding of biological processes and biomarker discovery. In this context, a proteomic analysis was carried out from plasma-EVs of an HIV/VL patient over two years and compared to HIV and healthy controls. The analysis confirmed classical EV markers but showed limited detection of Leishmania proteins. However, variations in human protein abundance related to relevant immunological processes were observed. Notably, the macrophage receptor with a collagenous structure (MARCO) was consistently detected only in the patient and not in the control groups. Significantly, the relevance of MARCO as a possible VL biomarker was confirmed using a validation cohort with five VL patients and its detection by Western Blot was possible. Although MARCO warrants further investigation as a VL related biomarker, the study of EVs confirmed their promise of being a privileged window into this disease. Future studies are needed to broaden data on EVs in infections to improve clinical management.

Introduction: Allogeneic hematopoietic stem cell transplant (HSCT) recipients remain highly susceptible to pneumococcal infection despite current vaccination strategies, and the contribution of T-cell–mediated immunity to protection in this population is not fully defined. Methods: We conducted a prospective study evaluating humoral and cellular immune responses to sequential pneumococcal vaccination with the 13-valent conjugate vaccine (PCV13) followed by the 23-valent polysaccharide vaccine (PPV23) in allogeneic HSCT recipients. Immune responses were assessed through serotype-specific antibody quantification, CD4 ⁺ T-cell proliferation assays, and cytokine profiling after in vitro stimulation with heat-killed Streptococcus pneumoniae . Results: Conjugate vaccination induced antigen-specific CD4 ⁺ T-cell proliferation and established T-cell memory. However, subsequent polysaccharide vaccination did not enhance CD4 ⁺ T-cell proliferation in sequentially vaccinated patients. PPV23 administration was associated with a decline in antibody titers for serotypes shared with the conjugate vaccine, while humoral responses to non-shared serotypes were preserved. Despite the lack of cellular boosting, the sequential schedule elicited a strong T-helper 17 cytokine response, characterised by increased secretion of interleukin-17A, interleukin-17F, and interleukin-22, suggesting activation of a pro-inflammatory pathway rather than expansion of functional immune memory. Discussion: This study provides, to our knowledge, the first integrated analysis of both humoral and T-cell immune responses to pneumococcal vaccination in allogeneic HSCT recipients, offering a translational perspective that links immunological mechanisms with clinical relevance. Our findings indicate that conjugate vaccination is essential for priming both cellular and humoral immunity, whereas polysaccharide boosting primarily broadens serotype coverage but may attenuate previously established immune responses. In the context of emerging higher-valency conjugate vaccines, including the recently introduced 21-valent formulation incorporating novel serotypes, these results support a reassessment of the need for polysaccharide boosters and inform optimisation of pneumococcal vaccination strategies in immunocompromised hosts at high risk for invasive disease.

Pteridine reductase 1 (PTR1) is a key folate pathway enzyme of pathogenic trypanosomatids that reduces biopterin to dihydro- and tetrahydrobiopterin. It is a promising target for drug design against diseases such as sleeping sickness or leishmaniasis. Amongst known PTR1 inhibitors, 2-aminobenzothiazole derivatives that target the PTR1 biopterin pocket were previously found to show good overall toxicity profiles and some of them display promising anti-parasite activity. On the other hand, compounds containing a 3,4-dichlorophenyl moiety, such as 1-(3,4-dichlorobenzyl)-1H-benzimidazol-2-amine (I), interact in a different region of the Trypanosoma brucei PTR1 (TbPTR1) binding pocket. In this work, we develop two compound series in which a 2-aminobenzothiazole moiety is connected to a 3,4-dichlorophenyl moiety via different linkers. A bespoke in-house compound library was virtually screened by computational docking against TbPTR1 and Leishmania major PTR1 (LmPTR1). Five selected compounds were synthesized, two of which, 1a and 1b, emerged as promising inhibitors of TbPTR1, with 1b also showing a micromolar inhibition level against LmPTR1 and moderate activity against Leishmania infantum (close to 50% at 10 µM), demonstrating its potential as a pan-parasite PTR1 inhibitor, which is rather unusual for 2-aminobenzothiazole derivatives. Quantum chemical calculations and crystallography guided the design of further compounds whose structure-activity relationship (SAR) showed that the halogen at the meta position of the phenyl ring is energetically more favorable for anti-PTR1 activity than at the para position, but that single halogen substitutions resulted in lower anti-parasite activities. Overall, our multidisciplinary fragment hybridization approach resulted in compounds with experimentally validated designed binding modes, that were less toxic and more active against the T. brucei parasite than the parent compound I.