Pathogens

16 pages, 1492 KB

Open AccessArticle

Characterization of the Gut Microbiome of Patients with Clostridioides difficile Infection and Healthy Individuals in Greece

by Dimitra Mougiou, Georgia Gioula, Lemonia Skoura, Fani Minti, Theodoros Karampatakis, Dimitrios Malandris, Konstantinos Pelekoudas and Melania Kachrimanidou

Pathogens 2026, 15(3), 275; https://doi.org/10.3390/pathogens15030275 (registering DOI) - 3 Mar 2026

Abstract

Background/Objectives: The gut microbiome plays an important role in the colonization of an individual by Clostridioides difficile and in the development of Clostridioides difficile infection (CDI). The main purpose of this study was to compare the gut microbiomes of patients with CDI and [...] Read more.

Background/Objectives: The gut microbiome plays an important role in the colonization of an individual by Clostridioides difficile and in the development of Clostridioides difficile infection (CDI). The main purpose of this study was to compare the gut microbiomes of patients with CDI and healthy individuals. Methods: We prospectively included 48 individuals: 32 patients with CDI and 16 healthy individuals. Microbiomes were analyzed by sequencing the hypervariable regions of the 16S rRNA gene using an Ion GeneStudio™ S5 System. Further statistical analysis of microbiome data was performed with the open-source programming language R version 3.5.2. Results: Among the CDI patients, Firmicutes and Proteobacteria were the most abundant phyla, while Enterobacteriaceae and Enterococcaceae were the most abundant families. Genus-level analysis showed that Enterococcus was the dominant genus in CDI patients; in contrast, in healthy individuals, Faecalibacterium was the most abundant. The MaAsLin2 tool revealed that members of the family Enterococcaceae and the genus Enterococcus were more abundant in patients with CDI than in healthy individuals. Alpha and beta diversity did not reveal differences between the two study groups. Conclusions: We observed differences in microbiome patterns between healthy individuals and CDI patients that were consistent with the literature. Further studies are needed. Full article

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17 pages, 1142 KB

Open AccessArticle

Prevalence, Virulence Determinants, and Antimicrobial Resistance of Staphylococcus aureus and Escherichia coli Isolated from Computer Devices Used by Staff and Students at a Northern Thailand University

by Sorawit Upakut, Achiraya Siriphap, Ornampai Japa, Pathumwan Watsing, Peerapat Bunpak, Aacharaporn Ta-In, Cholthicha Inmanee, Chutamas Thepmalee, Nittiya Suwannasom and Krissana Khoothiam

Pathogens 2026, 15(3), 274; https://doi.org/10.3390/pathogens15030274 - 3 Mar 2026

Abstract

Computer devices in university settings are frequently shared and repeatedly handled, making them potential reservoirs for pathogenic bacteria. This study aimed to investigate the prevalence, virulence determinants, and antimicrobial resistance profiles of Staphylococcus aureus and Escherichia coli isolated from computer devices used by [...] Read more.

Computer devices in university settings are frequently shared and repeatedly handled, making them potential reservoirs for pathogenic bacteria. This study aimed to investigate the prevalence, virulence determinants, and antimicrobial resistance profiles of Staphylococcus aureus and Escherichia coli isolated from computer devices used by staff and students at a university in Northern Thailand. A total of 400 computer devices were sampled, with each device defined as a single sampling unit comprising both the keyboard and computer mouse. Bacterial identification was performed using PCR, while staphylococcal enterotoxin (se) genes and diarrheagenic E. coli (DEC)-associated virulence genes were detected by PCR. Antimicrobial susceptibility was assessed using the disk diffusion method. Overall, 74 (18.5%) S. aureus isolates and 6 (1.5%) E. coli isolates were recovered. The highest prevalence of S. aureus was observed among personal-use student computer devices (29%; p < 0.001), whereas E. coli was most frequently detected on public-use staff computer devices (4%). Among S. aureus isolates, 24.3% (18/74) carried at least one se gene, with sec being the most prevalent (13.5%). Half of the E. coli isolates harbored the astA gene. Low resistance rates (<10%) were observed among S. aureus; however, four isolates (5.4%) were classified as MRSA, three of which exhibited multidrug resistance. All E. coli isolates were resistant to ampicillin, and 50% displayed multidrug-resistant phenotypes. These findings suggest that computer devices can act as occasional reservoirs of potentially pathogenic and antimicrobial-resistant bacteria in university environments. Full article

(This article belongs to the Special Issue Advanced Antimicrobial Agents: Combatting Multi-Drug Resistant Bacterial Infections)

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21 pages, 854 KB

Open AccessReview

Trypanosoma vivax in Water Buffaloes (Bubalus bubalis): A Host-Centered Synthesis of Pathogenesis, Epidemiology, Diagnosis, and Integrated Control with Implications for Tropical Production Systems

by André de Medeiros Costa Lins, Dryelle Vieira de Oliveira Brandão, Fernanda Monik Silva Martins, Aline Maia Silva, Henrique dos Anjos Bonjardim and Felipe Masiero Salvarani

Pathogens 2026, 15(3), 273; https://doi.org/10.3390/pathogens15030273 - 3 Mar 2026

Abstract

Trypanosoma vivax is a hemoprotozoan parasite of major veterinary importance affecting domestic ungulates in Africa and the Americas. While traditionally addressed within cattle-centered paradigms, accumulating evidence indicates that water buffaloes (Bubalus bubalis) are both clinically susceptible and epidemiologically significant hosts. This [...] Read more.

Trypanosoma vivax is a hemoprotozoan parasite of major veterinary importance affecting domestic ungulates in Africa and the Americas. While traditionally addressed within cattle-centered paradigms, accumulating evidence indicates that water buffaloes (Bubalus bubalis) are both clinically susceptible and epidemiologically significant hosts. This structured narrative review provides a host-centered synthesis of global evidence on T. vivax infection in buffaloes, integrating pathogenesis, transmission biology, epidemiology, diagnostics, chemotherapy, and integrated control. The analysis encompasses literature from 2000 to 2025 and incorporates seminal experimental studies published prior to 2000 that established buffalo susceptibility and reservoir competence. Evidence from cyclical (tsetse-mediated) and mechanical transmission systems is comparatively interpreted to clarify host–parasite dynamics. The Amazon biome is discussed as a model system for high-density buffalo production under mechanical vector pressure, offering case-based contextualization without geographic restriction. Particular attention is given to immunopathological mechanisms, chronic low-parasitemia carriage, diagnostic sensitivity in subclinical infections, emerging trypanocide resistance, and ecological constraints on vector control. Controversies and buffalo-specific knowledge gaps are highlighted throughout. By adopting a buffalo-centered analytical framework, this review supports translational diagnostics, targeted surveillance, and sustainable control strategies for trypanosomiasis in tropical livestock systems. Full article

(This article belongs to the Topic Advances in Infectious and Parasitic Diseases of Animals)

52 pages, 8288 KB

Open AccessArticle

Beyond the Mutation Abyss: Revisiting SARS-CoV-2 Receptor-Binding Domain Evolution from ACE2 Binding Optimization to Immune Epitope Remodeling

by Omar A. Soliman, Yasmine Shahine, Daniel Baecker and Ahmed Noby Amer

Pathogens 2026, 15(3), 272; https://doi.org/10.3390/pathogens15030272 - 3 Mar 2026

Abstract

The SARS-CoV-2 Omicron variant and its descendants accumulated unprecedented numbers of spike substitutions yet remained transmissible, implying compensatory mechanisms that preserve entry while eroding humoral immunity. We analyzed 32 variants for sequence-level mutation, physicochemical profiling, and epitope disruption; 25 had growth-advantage estimates, and [...] Read more.

The SARS-CoV-2 Omicron variant and its descendants accumulated unprecedented numbers of spike substitutions yet remained transmissible, implying compensatory mechanisms that preserve entry while eroding humoral immunity. We analyzed 32 variants for sequence-level mutation, physicochemical profiling, and epitope disruption; 25 had growth-advantage estimates, and 18 underwent molecular dynamics/MM-PBSA simulations. We applied a systems-virology framework to the SARS-CoV-2 receptor-binding domain (RBD), integrating immunodominance-weighted epitope conservation (567 B-cell and 97 T-cell epitopes) across variants (Wuhan-Hu-1 to KP.3) with molecular dynamics, molecular mechanics Poisson–Boltzmann surface area (MM-PBSA) binding energetics, and deep mutational scanning (DMS) benchmarking. B-cell epitope conservation declined from a median of 72.7% in pre-Omicron variants to 28.8% in BA.1 and 10.6% in KP.3, and was strongly inversely associated with a breakthrough-infection proxy (Spearman ρ = −0.8246, p < 0.001), whereas RBD T-cell epitopes remained comparatively conserved (91.5% to 87.2%). Despite the loss of the ancestral K417–ACE2 D30 salt bridge, Omicron reconfigured the interface via alternative electrostatic contacts (Q493R–E35 and Q498R–D38), producing compensatory interactions captured by MM-PBSA, but with only modest agreement with DMS affinity changes (r = 0.682, p = 0.007), consistent with enthalpy–entropy compensation. Finally, mutation tolerance shifted toward stronger epistatic buffering in Omicron (two-fold higher epistasis than pre-Omicron; p = 0.0093), enabling extensive antigenic change without structural collapse. Together, these results support a multi-objective evolutionary strategy—epitope erosion, interface rewiring, and epistatic compensation—that can be operationalized to prioritize emerging lineages for surveillance and to inform vaccine designs that emphasize conserved T-cell targets. Full article

(This article belongs to the Special Issue Antimicrobial Resistance in the Post-COVID Era: A Silent Pandemic)

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16 pages, 2188 KB

Open AccessArticle

Effect of trans-Cinnamaldehyde on Adhesion and Other Virulence Factors of Methicillin-Resistant Staphylococcus aureus

by Barbara Kot, Kamila Wierzchowska, Agata Grużewska, Elżbieta Anna Trafny, Małgorzata Stępińska and Małgorzata Witeska

Pathogens 2026, 15(3), 271; https://doi.org/10.3390/pathogens15030271 - 3 Mar 2026

Abstract

Methicillin-resistant Staphylococcus aureus (MRSA) produces virulence factors and causes hard-to-treat infections. This study aimed to evaluate the effect of trans-cinnamaldehyde (TC) on the selected virulence factors of MRSA: adhesion to host plasma and extracellular matrix proteins, protease, DNase and esterase production, and [...] Read more.

Methicillin-resistant Staphylococcus aureus (MRSA) produces virulence factors and causes hard-to-treat infections. This study aimed to evaluate the effect of trans-cinnamaldehyde (TC) on the selected virulence factors of MRSA: adhesion to host plasma and extracellular matrix proteins, protease, DNase and esterase production, and hemolytic activity. Our results showed that TC at ½ MBIC (Minimum Biofilm Inhibition Concentration) of 240 µg/mL or 60 µg/mL, depending on the isolate, significantly reduced MRSA adhesion. Inhibition varied between isolates, ranging from 26.1% to 41.3% (fibrinogen), 18.2% to 34.9% (elastin), 26.5% to 32.4% (laminin), and 17.1% to 30.5% (collagen). TC at ½ MIC (Minimum Inhibitory Concentration) of 30 µg/mL also significantly inhibited MRSA enzyme production, and reduced hemolytic activity (by 80.0–83.1%, depending on the isolate). TC may be an alternative to antibiotics for combating infections caused by S. aureus, as it not only reduces bacterial survival in the host but also reduces S. aureus virulence at subinhibitory concentrations. TC at higher concentrations exhibits cytotoxicity in human fibroblasts, limiting its topical use. Therefore, to exploit TC’s antibacterial potential, it is necessary to identify substances that act synergistically with it, enabling reduced effective doses. Full article

(This article belongs to the Special Issue Challenges of Multidrug-Resistant Bacteria and Drug Development of Antibacterials)

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20 pages, 900 KB

Open AccessReview

Plastic-Derived Pollutants as Emerging Modifiers of Viral Diseases

by Fatima Hisam, Ramina Kordbacheh, Ebenezer Senu, Spandan Mukherjee, Jon Sin and Erica L. Sanchez

Pathogens 2026, 15(3), 270; https://doi.org/10.3390/pathogens15030270 (registering DOI) - 3 Mar 2026

Abstract

Plastic pollutants, including phthalates, bisphenol A (BPA), per- and polyfluoroalkyl substances (PFAS), and microplastics (MPs), are increasingly recognized as emerging environmental cofactors that intersect with infectious disease dynamics. These compounds, once considered inert, can alter immune function, reshape host–pathogen interactions, and directly influence [...] Read more.

Plastic pollutants, including phthalates, bisphenol A (BPA), per- and polyfluoroalkyl substances (PFAS), and microplastics (MPs), are increasingly recognized as emerging environmental cofactors that intersect with infectious disease dynamics. These compounds, once considered inert, can alter immune function, reshape host–pathogen interactions, and directly influence viral survival and transmission. In this review, we compile current evidence on the chemistry, environmental occurrence, and biological activity of major plastic-associated pollutants with emphasis on their role in viral infections. Phthalates such as di(2-ethylhexyl) phthalate (DEHP) and its metabolite MEHP modulate innate immune signaling and have been shown to exacerbate infections, including Dengue and Coxsackievirus B3. Other DEHP-like phthalates, such as dibutyl phthalate (DBP), exhibit consistent infection-enhancing effects, while high molecular weight or cyclical phthalates such as polyvinyl acetate phthalate (PVAP) display conflicting results in their modulation of viral infections. BPA, widely detected in human tissues, acts through endocrine and immune disruption, worsening viral myocarditis, and altering influenza outcomes. PFAS, persistent “forever chemicals,” reshape adaptive immune responses and are associated with increased susceptibility, viral persistence, or severity of infection of herpesvirus (HCMV, EBV, HSV-1), hepatitis virus, and influenza infection. Microplastics represent a distinct risk by acting as physical carriers for viruses and bacteria, stabilizing viral RNA, enhancing host cell uptake, and skewing immune responses. Together, these pollutants extend beyond toxicology into virology, providing novel insights into how environmental exposures converge with viral pathogenesis. We highlight mechanistic advances and critical knowledge gaps and propose future directions for integrating environmental health and infectious disease research. Full article

(This article belongs to the Section Viral Pathogens)

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13 pages, 2203 KB

Open AccessArticle

Pro-Inflammatory Response of Bovine Lung Explant Induced by Mycoplasma mycoides subsp. mycoides

by Leruo Keokilwe, Giovanni Di Teodoro, Marta Di Federico, Massimo Ancora, Ivanka Krasteva, Gianluca Orsini, Cesare Camma, Fabrizia Perletta, Chiara Di Pancrazio, Mirella Luciani, Chandapiwa Marobela-Raborokgwe, Massimo Scacchia and Flavio Sacchini

Pathogens 2026, 15(3), 269; https://doi.org/10.3390/pathogens15030269 - 3 Mar 2026

Abstract

Contagious bovine pleuropneumonia (CBPP) is a significant respiratory disease in cattle caused by Mycoplasma mycoides subsp. mycoides (Mmm). A better understanding of the pathogenesis of CBPP and the immune response of the host to infection will assist in the development of [...] Read more.

Contagious bovine pleuropneumonia (CBPP) is a significant respiratory disease in cattle caused by Mycoplasma mycoides subsp. mycoides (Mmm). A better understanding of the pathogenesis of CBPP and the immune response of the host to infection will assist in the development of novel interventions to prevent disease progression. In this study, bovine lung explants (BLEs) were exposed to Mmm to investigate the upregulation and release of early inflammatory cytokines, mediators and receptors following tissue infection. Immunomodulatory molecules indicative of cell activation were investigated by immunoblotting on the BLEs and the tissue culture supernatants, and quantitative real-time PCR (RTq-PCR) was performed on the BLEs to determine the fold change in the expression of the respective mRNA. Immunoblotting indicated the production of inflammatory cytokines, mediators and receptors in Mmm-infected BLEs; however, this contrasted strongly with the mRNA expression profile, which did not show any significant fold increase. Infection of the BLEs with Mmm stimulated the production of some pro-inflammatory cytokines and mediators, including IL-1β, COX-2, 5-LOX and iNOS. Toll-like receptor proteins TLR2 and TLR4 were detected solely in the tissue culture supernatant of Mmm-infected BLEs. These receptors are considered to be involved in the recognition of Mmm by BLE tissue cells, thus triggering intracellular pathways that produce specific inflammatory cytokines and mediators, initiating the inflammatory response. Full article

(This article belongs to the Special Issue Mycoplasmas in Respiratory Tract Infections of Cattle: 2nd Edition)

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10 pages, 663 KB

Open AccessArticle

Acomys cahirinus seurati as a Potential Reservoir Host of Leishmania major

by Sergei Karlin, Barbora Bečvářová, Kamal Eddine Benallal, Tomáš Bečvář, Ghania Mezai, Mounir Zaabar, Mohamed Mefissel, Petr Volf and Jovana Sádlová

Pathogens 2026, 15(3), 268; https://doi.org/10.3390/pathogens15030268 - 3 Mar 2026

Abstract

Cutaneous leishmaniasis (CL) caused by Leishmania major is a zoonotic disease transmitted by phlebotomine sand flies. Identification of reservoir hosts is critical for understanding transmission and guiding control. While Psammomys obesus and Meriones shawi are recognized reservoirs in North Africa, the role of [...] Read more.

Cutaneous leishmaniasis (CL) caused by Leishmania major is a zoonotic disease transmitted by phlebotomine sand flies. Identification of reservoir hosts is critical for understanding transmission and guiding control. While Psammomys obesus and Meriones shawi are recognized reservoirs in North Africa, the role of other rodents remains unclear. We experimentally assessed the reservoir potential of Acomys cahirinus seurati from southeastern Algeria. Animals were intradermally infected, and clinical signs and infectiousness to sand flies were monitored. Parasite persistence in skin and visceral tissues was measured by quantitative PCR. All animals developed localized, self-limiting lesions. Seven of sixteen individuals transmitted parasites to Phlebotomus papatasi between 10 and 25 weeks post-infection. Parasites were mostly restricted to the inoculation site, with limited dissemination to contralateral pinnae and hindpaws, and rare presence in spleen or liver. Notably, some animals were infectious without visible lesions, indicating that ulceration is not required for transmission. These findings show that A. cahirinus seurati can maintain L. major infection for up to 6 months and transmit parasites to sand flies, fulfilling key criteria of a reservoir host. Thus, this species may contribute to CL transmission in endemic foci lacking classical reservoirs, emphasizing the need to consider alternative rodent hosts in surveillance and control programs in North Africa. Full article

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15 pages, 2081 KB

Open AccessCommunication

Characterization of Salmonella spp. Based on CRISPR PCR and FT-IR Approaches: A Pilot Study in Northern Italy

by Clara Tramuta, Irene Floris, Monica Pitti, Giulia Federica Cazzaniga, Miriam Cordovana and Daniela Manila Bianchi

Pathogens 2026, 15(3), 267; https://doi.org/10.3390/pathogens15030267 - 2 Mar 2026

Abstract

The aim of the present study was to employ a genotypic method (CRISPR PCR) and a phenotypic method (FT-IR combined with Linear Discriminant Analysis, LDA) for the rapid identification and discrimination of major virulent Salmonella serovars. Specifically, a total of 24 Salmonella Enteritidis, [...] Read more.

The aim of the present study was to employ a genotypic method (CRISPR PCR) and a phenotypic method (FT-IR combined with Linear Discriminant Analysis, LDA) for the rapid identification and discrimination of major virulent Salmonella serovars. Specifically, a total of 24 Salmonella Enteritidis, 24 Salmonella Typhimurium, 24 S. Typhimurium 4,5,12:i:-, and 14 Salmonella Infantis strains, previously serotyped according to the Kaufmann–White scheme, were analyzed. CRISPR PCR generated serotype-specific amplicons: 850 bp for S. Enteritidis, 700 bp and 2000 bp for S. Typhimurium, 1300 bp and 1500 bp for S. Typhimurium 4,5,12:i:-, and 1000 bp and 1900 bp for S. Infantis. FT-IR spectra of all serotypes were analyzed by LDA, which revealed a clear separation of three serotype-defined groups: S. Enteritidis, S. Infantis, and a group comprising S. Typhimurium and its monophasic variant 4,5,12:i:-. Further discrimination based on CRISPR-defined classes was observed within each serovar: two classes for S. Typhimurium, two for S. Typhimurium 4,5,12:i:-, and three for S. Infantis. Overall, the combined CRISPR PCR and FT-IR-LDA approach provides preliminary evidence supporting the potential application of a combined approach for the epidemiological surveillance of Salmonella. Full article

(This article belongs to the Special Issue Advances in Salmonella Epidemiology and Pathogenesis)

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23 pages, 2214 KB

Open AccessArticle

Identification of TgENT1 as the TgUUT1 Uracil/Uridine Transporter of Toxoplasma gondii

by Hamza A. A. Elati, Mariana Ferriera Silva, Lilach Sheiner and Harry P. de Koning

Pathogens 2026, 15(3), 266; https://doi.org/10.3390/pathogens15030266 - 2 Mar 2026

Abstract

The protozoan pathogen Toxoplasma gondii is responsible for toxoplasmosis, a disease that can be deadly in immunocompromised patients and the developing fetus during pregnancy. Current treatments are widely considered to be suboptimal. We have recently reported that 5-fluoropyrimidines have highly promising anti-toxoplasmosis effects [...] Read more.

The protozoan pathogen Toxoplasma gondii is responsible for toxoplasmosis, a disease that can be deadly in immunocompromised patients and the developing fetus during pregnancy. Current treatments are widely considered to be suboptimal. We have recently reported that 5-fluoropyrimidines have highly promising anti-toxoplasmosis effects and are internalized by the parasite by a high-affinity uracil/uridine transporter, TgUUT1. Here, we attempt to identify the gene encoding this transport protein. The only nucleoside or nucleobase family identified in the T. gondii genome was the Equilibrative Nucleoside Transporter (ENT) family, with four members. Of these, TgAT1 is known to be purine-specific, and deletion of the TgENT2 and TgENT3 genes, either separately or jointly, did not affect uridine transport or sensitivity to 5-fluoropyrimidines. In contrast, depletion of TgENT1, an essential gene, resulted in a significant reduction in the uptake of both uracil and uridine but not of the amino acid tryptophan. Moreover, expression of TgENT1 in a Leishmania mexicana cell line with low endogenous uracil uptake rates significantly increased uracil uptake for these cells. We conclude that it is highly probable that TgENT1 encodes the T. gondii uracil/uridine transporter. On the basis of our previous results, we infer that TgENT1 likely also mediates the uptake of 5-fluoropyrimidines. Full article

(This article belongs to the Special Issue Toxoplasmosis in Humans and Animals)

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21 pages, 3192 KB

Open AccessArticle

The Exploration of Therapeutic Antivirals for Human Papillomavirus in the Last 40 Years: Bibliometric Research

by Zixiao Jiang, Liangrui Jin, Chengjun Wu, Zhenqing Li, Zhangrong Lou and Peng Qu

Pathogens 2026, 15(3), 265; https://doi.org/10.3390/pathogens15030265 - 2 Mar 2026

Abstract

Human papillomavirus (HPV) is a well-known carcinogenic DNA virus, responsible for about 4% of all cancer cases globally. Effective antiviral treatments for those who are already infected with HPV are still in their early stages, despite the fact that prophylactic vaccinations have shown [...] Read more.

Human papillomavirus (HPV) is a well-known carcinogenic DNA virus, responsible for about 4% of all cancer cases globally. Effective antiviral treatments for those who are already infected with HPV are still in their early stages, despite the fact that prophylactic vaccinations have shown impressive success in preventing new infections. Effective treatments for HPV-related malignancies are also hampered by the fact that current articles address a wide spectrum of pathways but lack thorough systematic studies. In this work, we use bibliometric techniques to examine research trends and innovative approaches in the development of HPV antivirals over the last 40 years. Our results are intended to offer insightful information and direct future research into effective antiviral treatments for HPV-induced cancers. Full article

(This article belongs to the Special Issue Epidemiology, Diagnostics, and Therapeutics of High-Risk Human Papillomavirus (HPV) Infections)

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19 pages, 1701 KB

Open AccessArticle

Integrated Laboratory Evaluation of Rift Valley Fever Virus Antibodies Using the Competitive ELISA and Virus Neutralization Test

by Ommer M. Dafalla, Abdullah A. Alashor, Mohammed O. Hussien, Elsiddig M. Noureldin, Tellal B. Ageep, Mohammed A. Najmi, Mohamed S. Mohamed, Ali A. Hakami, Saleh A. Alrashedi, Fisal A. Bushlaibi and Fahad N. Abukhalil

Pathogens 2026, 15(3), 264; https://doi.org/10.3390/pathogens15030264 - 2 Mar 2026

Abstract

Background: Rift Valley fever virus (RVFV) is a significant mosquito-borne zoonotic virus with high public health and veterinary importance in Africa and the Middle East. Reliable diagnostic assays for detecting antibodies and assessing their functional neutralizing capacity are essential for surveillance programs, vaccine [...] Read more.

Background: Rift Valley fever virus (RVFV) is a significant mosquito-borne zoonotic virus with high public health and veterinary importance in Africa and the Middle East. Reliable diagnostic assays for detecting antibodies and assessing their functional neutralizing capacity are essential for surveillance programs, vaccine monitoring, and outbreak preparedness. Objective: This study evaluates and compares the analytical performance of a competitive enzyme-linked immunosorbent assay (cELISA) and a virus neutralization test (VNT) for detecting RVFV antibodies in vaccinated sheep sera, establishing an integrated laboratory workflow for virus titration, serological detection, and functional neutralization. Methods: Twenty serum samples were collected from sheep pre-vaccination and one month post-vaccination with Smithburn live attenuated RVFV vaccine. Sera were tested using a commercial multispecies RVFV competitive ELISA to detect antibodies specific to the viral nucleocapsid protein. Viral titration was conducted in Vero cells, and 50% tissue culture infective dose (TCID₅₀/0.1 mL) was calculated using the Reed and Muench method. VNT was performed at 24, 48, 72, and 96 h after infection with different viral doses (10² to 10⁵ TCID₅₀/0.1 mL), and the neutralizing ability of serial serum dilutions (1:2 to 1:1024) was tested. Compared with the control, protection was determined by cytopathic effect (CPE) inhibition. Results: ELISA revealed robust antibody signals up to a 1:32 dilution, with signal-to-noise (S/N) < 40%, whereas for higher dilutions, antibody detection became inconclusive or negative. Virus titration was performed to verify a stock concentration of 10^6.5 TCID₅₀/0.1 mL. The VNT exhibited time- and dose-dependent kinetics; high protection rates (≥97) were observed at 1:2–1:8 dilutions against 10²–10³ TCID₅₀/0.1 mL challenge doses; however, neutralizing efficacy decreased significantly at higher viral loads and higher serum dilutions. While cELISA and VNT results correlated strongly at low serum dilutions, the cELISA showed decreased sensitivity at dilutions ≥ 1:64, where the VNT remained capable of detecting functional neutralizing activity. Conclusions/Discussion: The results demonstrate that while both assays correlate well at high antibody concentrations, they diverge at lower concentrations. This discrepancy highlights the functional difference between binding antibodies (N-protein) and neutralizing antibodies (Gn/Gc glycoproteins). Consequently, the cELISA is ideal for rapid screening, whereas the VNT is indispensable for confirming functional immunity. Integrating both assays provides a more accurate immunological profile for RVFV surveillance and vaccine evaluation. Full article

(This article belongs to the Section Viral Pathogens)

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24 pages, 15884 KB

Open AccessArticle

Acanthamoeba castellanii: Non-Steroidal Anti-Inflammatory Drugs Affect Adhesion, Motility, and Encystment, Suggesting a Link with a gp63-like Protein Candidate

by Verónica I. Hernández-Ramírez, Hugo Varela-Rodríguez, Luis Varela-Rodríguez, Francisco Sierra-López, Daniela Eloísa San Juan-Mora, José Daniel Morales-Mora, Daniela Falcón-Navarrete, Carlos Osorio-Trujillo, Jacqueline Ríos-López, Itzel Berenice Rodríguez-Mera, María Maricela Carrasco-Yépez and Patricia Talamás-Rohana

Pathogens 2026, 15(3), 263; https://doi.org/10.3390/pathogens15030263 - 2 Mar 2026

Abstract

Acanthamoeba castellanii, an opportunistic free-living amoeba, causes severe infections including Acanthamoeba keratitis. This exploratory study evaluated whether three non-steroidal anti-inflammatory drugs (NSAIDs)—acetylsalicylic acid, ibuprofen, and diclofenac (100 µM)—modulate pathogenicity-related processes in A. castellanii and explored the involvement of a gp63-like protein during [...] Read more.

Acanthamoeba castellanii, an opportunistic free-living amoeba, causes severe infections including Acanthamoeba keratitis. This exploratory study evaluated whether three non-steroidal anti-inflammatory drugs (NSAIDs)—acetylsalicylic acid, ibuprofen, and diclofenac (100 µM)—modulate pathogenicity-related processes in A. castellanii and explored the involvement of a gp63-like protein during encystment and adhesion. Trophozoites were continuously exposed to each drug and analyzed for adhesion, migration on host-derived discontinuous brain micropatterns, encystment efficiency, and parasite-induced cytoskeletal remodeling in MDCK epithelial cells. In silico docking was performed to assess potential drug–protein interactions. Drug exposure reduced adhesion with maximal inhibition at 60 min. After 1 h, migration decreased by 49%, 64%, and 38%, and encystment was reduced by 50%, 85%, and up to 90%, respectively, in cultures treated with acetylsalicylic acid, ibuprofen, and diclofenac. Co-incubation with untreated trophozoites lowered actin fluorescence to approximately 50%, whereas drug-treated co-cultures preserved fluorescence near control levels. Colocalization analysis showed increased spatial overlap between gp63-like protein and F-actin in cysts (~40%) and migrating trophozoites (~20%) compared with non-stimulated forms (~3.8%). Collectively, these findings suggest that NSAID-sensitive pathways influence host interaction, migration, and encystment in A. castellanii and allow for the proposal of gp63-like protein as a putative molecular component of the NSAIDs sensitive pathways. Full article

(This article belongs to the Special Issue XXth International Meeting on the Biology and Pathogenicity of Free-Living Amoebae (FLAM 2025))

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17 pages, 12247 KB

Open AccessArticle

In Vivo Confocal Microscopy as a Prognostic Indicator in Acanthamoeba Keratitis: Insights from a Retrospective Study

by Yiping Han, Yuan Wei, Qiankun Chen, Jinding Pang, Qingquan Shi and Qingfeng Liang

Pathogens 2026, 15(3), 262; https://doi.org/10.3390/pathogens15030262 - 2 Mar 2026

Abstract

Background: To assess longitudinal changes in the in vivo confocal microscopy (IVCM) features during Acanthamoeba keratitis (AK) treatment and develop a prognostic model. Methods: This retrospective study included 59 AK patients who underwent IVCM at baseline and 1 and 3 months. Fourteen morphological [...] Read more.

Background: To assess longitudinal changes in the in vivo confocal microscopy (IVCM) features during Acanthamoeba keratitis (AK) treatment and develop a prognostic model. Methods: This retrospective study included 59 AK patients who underwent IVCM at baseline and 1 and 3 months. Fourteen morphological features covering pathogen-related characteristics, cyst arrangement patterns, and inflammatory markers were compared between good and poor prognosis groups, which were defined based on clinical outcomes including corneal perforation, the need for therapeutic keratoplasty, or final best-corrected visual acuity (BCVA) ≤ 0.05. Prognostic modeling was performed exclusively using baseline IVCM features and applied univariable and Firth-corrected multivariable logistic regression with collinearity assessment and clinical filtering, followed by 5-fold cross-validation. Results: Among 59 AK patients, 45 (76.3%) had a good prognosis and 14 (23.7%) had a poor prognosis. Poor prognosis eyes showed a higher prevalence of double-walled cysts, trophozoites, and clustered cysts, along with higher cyst density and deeper stromal invasion. In contrast, good-prognosis eyes had more target-like cysts, immature dendritic cells, and mature dendritic cells. Clustered cysts independently predicted poor prognosis (OR = 2.98), whereas target-like cysts (OR = 0.26) and mature dendritic cells (OR = 0.37) were protective (AUC = 0.883; all p < 0.05). Conclusions: IVCM provides a quantitative tool for early outcome prediction and individualized management. Higher cyst burden, clustered cysts, and persistent stromal involvement indicated poorer prognosis, whereas target-like cysts and mature dendritic cells indicated better prognosis. Full article

(This article belongs to the Section Parasitic Pathogens)

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22 pages, 1671 KB

Open AccessArticle

Antimicrobial Resistance in Acinetobacter baumannii Isolated from Ready-to-Eat Foods in Saudi Arabia

by Eman Marzouk and Adil Abalkhail

Pathogens 2026, 15(3), 261; https://doi.org/10.3390/pathogens15030261 - 1 Mar 2026

Abstract

Acinetobacter baumannii is widely recognized as a problematic pathogen in healthcare settings due to its ability to acquire resistance to multiple antimicrobial agents. However, less attention has been given to its presence outside hospitals. In this cross-sectional, laboratory-based surveillance study, we investigated the [...] Read more.

Acinetobacter baumannii is widely recognized as a problematic pathogen in healthcare settings due to its ability to acquire resistance to multiple antimicrobial agents. However, less attention has been given to its presence outside hospitals. In this cross-sectional, laboratory-based surveillance study, we investigated the occurrence of A. baumannii in ready-to-eat (RTE) foods sold at retail outlets in four cities of the Al-Qassim region, Saudi Arabia, during a single season. A total of 240 RTE food samples were analyzed using culture-based and molecular approaches for species confirmation, and antimicrobial susceptibility profiles were determined. A. baumannii was identified in 19 samples (7.9%), spanning several food categories. Most isolates showed resistance to multiple antimicrobial classes, and 16 met the criteria for multidrug resistance (MDR). Among the confirmed isolates, bla_OXA-23-like was detected in 16 (84.2%), bla_{OXA-24/40-like} in 2 (10.5%), and bla_OXA-58-like in 1 (5.3%). Resistance to fluoroquinolones, tetracyclines, and aminoglycosides was common, and OXA-type carbapenemase genes were detected in 16 isolates. These findings indicate that RTE foods can represent non-clinical environments in which MDR A. baumannii may be detected. Including food sources in antimicrobial resistance surveillance may therefore strengthen our understanding of the ecology of this pathogen within a One Health framework. Full article

(This article belongs to the Special Issue Acinetobacter baumannii: An Emerging Pathogen)

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13 pages, 1149 KB

Open AccessArticle

New Insights into the Combined Antiviral Effect of Extracts from Nerium oleander and Boswellia sacra Against Respiratory Syncytial Virus: A Preliminary Report

by Rebecca Piras, Luca Virdis, Valeria Manca, Marta Cogoni, Vanessa Palmas, Matthew G. Donadu, Aldo Manzin, Giuseppina Sanna and Luay Rashan

Pathogens 2026, 15(3), 260; https://doi.org/10.3390/pathogens15030260 - 1 Mar 2026

Abstract

In recent years, the emergence of drug-resistant pathogens and the limitations of current therapies have highlighted the need for innovative strategies to combat emerging viral infections. Natural compounds, derived from plants, are playing an increasingly significant role in the research of novel and [...] Read more.

In recent years, the emergence of drug-resistant pathogens and the limitations of current therapies have highlighted the need for innovative strategies to combat emerging viral infections. Natural compounds, derived from plants, are playing an increasingly significant role in the research of novel and effective therapies. Boswellia sacra, a frankincense-producing tree widely distributed in Yemen and Oman, and Nerium oleander, a common ornamental and medicinal plant, are examples of plants with well-documented antimicrobial properties. Their extracts have demonstrated good activity against a wide range of infections, which is attributed to the anti-inflammatory and immunomodulatory compounds they contain. Based on these findings, we assessed, in vitro, the broad-spectrum antiviral activity of combined extracts obtained from Boswellia sacra and Nerium oleander. The extract mixture NOBS7(1) was found to be active against the respiratory virus RSV, Herpesvirus simplex type 1, and Coxsackievirus 5. Furthermore, a combination of cell-based assays was performed to provide additional insights into their potential mechanism of action. Full article

(This article belongs to the Special Issue Antiviral Strategies Against Human Respiratory Viruses)

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11 pages, 721 KB

Open AccessArticle

Assessment of Systemic Inflammation as a Tool for Estimating the Risk of Death by Visceral Leishmaniasis

by Ingridi de Souza Sene, Vladimir Costa Silva, Débora Cavalcante Brás, Dorcas Lamounier Costa, Gabriel Reis Ferreira and Carlos Henrique Nery Costa

Pathogens 2026, 15(3), 259; https://doi.org/10.3390/pathogens15030259 - 28 Feb 2026

Abstract

Background: Visceral leishmaniasis (VL) is a life-threatening protozoan disease prevalent in tropical and subtropical regions and a frequent coinfection among people living with HIV. Early identification of patients at high risk of death may reduce case-fatality. This study evaluated the post-test prognostic value [...] Read more.

Background: Visceral leishmaniasis (VL) is a life-threatening protozoan disease prevalent in tropical and subtropical regions and a frequent coinfection among people living with HIV. Early identification of patients at high risk of death may reduce case-fatality. This study evaluated the post-test prognostic value of C-reactive protein (CRP) and interleukin-6 (IL-6) as biomarkers of mortality in VL. Methods: A retrospective hospital-based cohort of 101 VL patients was analyzed. CRP and IL-6 concentrations at admission were correlated with clinical findings, the Kala-Cal^® prognostic score, and in-hospital mortality. Results: Eight patients died, most presenting with hemorrhagic manifestations. At admission, 87.1% of patients had both biomarkers above the predefined cut-offs. CRP and IL-6 levels were markedly elevated in patients with hemorrhage or fatal outcomes. The AUC was 0.85 for CRP and 0.87 for IL-6, with no significant difference between markers. Optimal prognostic cut-offs were 150 mg/L for CRP and 90 pg/mL for IL-6. Conclusions: In this sample, CRP and IL-6 showed good prognostic performance in VL. In patients with low initial clinical risk, positive biomarker results substantially increased the probability of death. When combined with Kala-Cal^®, these markers may improve risk stratification and guide referral decisions. Full article

(This article belongs to the Section Parasitic Pathogens)

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Graphical abstract

11 pages, 554 KB

Open AccessArticle

Rift Valley Fever Virus Exposure in Camels and Horses Across Northern Nigeria Livestock Markets

by David Odion Ehizibolo, Olumuyiwa Oyekan, Nicodemus Mkpuma, Habibu Haliru, Ibrahim Garba, Isa Zayyad Turaki, Elizabeth Ene Williams, Agom Danmarwa, Abdullahi Mohammed, Musa Abdullahi Muhammad, Mansur Abubakar, Corrie Brown and Bonto Faburay

Pathogens 2026, 15(3), 258; https://doi.org/10.3390/pathogens15030258 - 28 Feb 2026

Abstract

Rift Valley Fever (RVF) is a neglected vector-borne zoonotic disease of significant veterinary and public health concern in Sub-Saharan Africa. This study investigated the seroprevalence of Rift Valley Fever Virus (RVFV) exposure and associated risk factors among camels and horses marketed in northern [...] Read more.

Rift Valley Fever (RVF) is a neglected vector-borne zoonotic disease of significant veterinary and public health concern in Sub-Saharan Africa. This study investigated the seroprevalence of Rift Valley Fever Virus (RVFV) exposure and associated risk factors among camels and horses marketed in northern Nigeria. A total of 1117 animals were sampled, comprising camels (812) and horses (305), across three major livestock markets (Maigatari, Maiduguri, and Illela). The overall seroprevalence was 18.8% (95% CI: 16.6–21.2%), with a striking six-fold disparity: camels showed a prevalence of 24.4% (95% CI: 21.6–27.4%), while horses exhibited only 3.9% (95% CI: 2.1–7.0%). Significant geographic clustering was observed, with Illela camels recording the highest prevalence (34.8%) compared to those in Maigatari (20.3%) and Maiduguri (20.2%). There were no significant associations with age or sex among camels. However, in horses, females were significantly more likely to test positive than males (OR = 0.27, 95% CI: 0.07–0.97). These findings demonstrate endemic RVFV circulation in Nigerian livestock, highlighting species- and location-specific differences, and underscore the zoonotic risks within regional and transboundary livestock trade networks. Full article

(This article belongs to the Section Emerging Pathogens)

14 pages, 8034 KB

Open AccessArticle

Dynamics of Interleukin-9 Producing Lymphocytes in Strongyloides ratti-Infected Mice

by Wiebke Hartmann, Lennart Heepmann, Lara Linnemann, Paula Licona-Limon, Florent Colomb, Tania Frangova, Henry J. McSorley and Minka Breloer

Pathogens 2026, 15(3), 257; https://doi.org/10.3390/pathogens15030257 - 28 Feb 2026

Abstract

Helminths infect a quarter of the human population and are controlled in the frame of a canonical type-2 immune response. Interleukin-9 is a cytokine with pleiotropic functions during type-2 immunity that can be produced by many different cells. Accumulating evidence suggest that IL-9 [...] Read more.

Helminths infect a quarter of the human population and are controlled in the frame of a canonical type-2 immune response. Interleukin-9 is a cytokine with pleiotropic functions during type-2 immunity that can be produced by many different cells. Accumulating evidence suggest that IL-9 is of particular relevance in controlling intestinal helminth infections. Using mice infected with the parasitic nematode Strongyloides ratti, we showed previously that ejection from the intestine depends on IL-9 and IL-9-mediated activation of mucosal mast cells. Here we use IL-9 reporter mice to identify the relevant cellular sources of IL-9 in vivo. We report that predominantly CD4⁺ T cells and group 2 innate lymphoid cells (ILC2s) produced IL-9 in S. ratti-infected or IL-33-treated mice. Interestingly, the IL-33-mediated induction of IL-9 and subsequent mast cell degranulation was modulated by concurrent S. ratti infection. While the IL-33-mediated expansion of IL-9-producing ILC2s was supressed by S. ratti infection, IL-9-producing CD4⁺ T cells were proportionally increased. Finally, we show that S. ratti-derived E/S products interfered with IL-9 production by BM-derived ILC2 in vitro. In conclusion, we have identified that ILC2 and CD4⁺ T cells produce IL-9 during S. ratti infection, and that ILC2 responses are suppressed by S. ratti products. Full article

(This article belongs to the Special Issue Immunology Insights: How the Immune System Battles Parasitic Infections)

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