Pathogens doi: 10.3390/pathogens15050492

Authors: Ixchel Salter Michaele-Francesco Corbisiero Daniel B. Chastain Chia-Yu Chiu Leland Shapiro Jose G. Montoya Raymund R. Razonable Andrés F. Henao-Martínez

Cytomegalovirus (CMV) establishes lifelong latency following primary infection and can reactivate to cause severe illness in immunocompromised hosts. CMV DNAemia in non-HIV-infected, non-solid organ/bone marrow transplant (NHNT) hosts is poorly characterized, with limited clinical insights. We aim to describe the clinical presentation, prognostic indicators, and outcomes of CMV DNAemia among NHNT patients. We used the TriNetX international patient database to identify adult patients diagnosed with CMV DNAemia from 2016 until March 2023. We evaluated hospitalization, intensive care unit (ICU) level care, and all-cause mortality at 30 days and 1 year. We also completed a post-propensity score analysis comparing clinical characteristics of survivors versus non-survivors at 90 days. We identified 1123 NHNT patients with CMV DNAemia, most of whom had neoplasms (63%). Venous thromboembolism occurred in 31% of the population. The 30-day hospitalization and all-cause mortality rates were 35% and 14%, respectively. After propensity score matching and Bonferroni correction, weakness, purpura, acute respiratory failure, malnutrition, encephalopathy, and hypotension were associated with increased 90-day all-cause mortality. NHNT patients with CMV DNAemia carry a substantial morbidity and all-cause mortality. Further studies are warranted to clarify whether CMV DNAemia is a causative factor or an incidental finding in this population.

Pathogens doi: 10.3390/pathogens15050491

Authors: Su Yeon Kim Taek-Kyun Lee Tae-Jin Choi

RNA viruses exhibit high mutation rates, necessitating antivirals targeting conserved infection mechanisms. In this study, viral hemorrhagic septicemia virus (VHSV), a non-human pathogenic negative-sense RNA virus, was used as a surrogate model to enable high-throughput antiviral screening under reduced biosafety conditions. A recombinant VHSV expressing enhanced green fluorescent protein was used to screen 17,265 compounds, 2000 plant extracts, and 100 marine extracts. Among the candidates, the leaf extract of Phellodendron amurense Rupr. (PL extract) exhibited antiviral activity with low cytotoxicity (selectivity index ≈ 10). The extract reduced viral infectivity in a dose-dependent manner and showed cross-activity against snakehead rhabdovirus. Mechanistic analyses indicated that the PL extract acts primarily at early stages of infection. Virucidal assays demonstrated direct, time-dependent inactivation of viral particles, while pre-treatment reduced host cell susceptibility. Time-of-addition experiments confirmed that antiviral activity was restricted to early infection, suggesting interference with viral attachment or entry rather than intracellular replication. Fractionation revealed that activity was associated with the non-polar n-hexane fraction, implicating lipophilic compounds that may disrupt viral envelope integrity or membrane interactions. These findings suggest that P. amurense leaf extract is a promising candidate for broad-spectrum antivirals targeting conserved entry processes in enveloped RNA viruses.

Pathogens doi: 10.3390/pathogens15050490

Authors: Meng Ling Moi

Functional antibody-dependent enhancement (ADE) represents a fundamental and context-dependent characteristic of antiviral antibody responses, reflecting the dual capacity of antibodies to mediate both the neutralization and Fc receptor-dependent enhancement of infection. In flavivirus research, this duality complicates the interpretation of conventional serological metrics and limits the reliability of single-parameter correlates of immunity, particularly in populations with complex exposure histories. Over the past decade, functional ADE assays have evolved from specialized mechanistic tools into integrated immune assessment platforms supporting translational immunology, vaccine evaluation, and population-level immune surveillance. These platforms incorporate Fcγ receptor-relevant target cell systems, standardized viral inputs, dilution series-based profiling, quantitative enhancement metrics, and structured quality control frameworks to enable reproducible, comparable, and context-aware functional measurements across cohorts and laboratories. A central concept emerging from these developments is that ADE reflects a dynamic functional immune state rather than an intrinsic property of antibodies or a direct indicator of pathological risk. Accordingly, functional ADE platforms support the contextual interpretation of antibody activity across physiologically relevant conditions, facilitating discrimination between transient functional enhancement and clinically meaningful immunological risk. By integrating functional ADE metrics with serological, cellular, and epidemiological data, these platforms provide a structured framework for interpreting immune profiles in vaccine evaluation, booster strategy design, and population-level risk stratification. This review synthesizes the development, standardization, and global dissemination of functional ADE platforms and discusses key principles governing biological relevance, analytical robustness, and inter-site transferability. Emerging directions integrating functional ADE profiling with systems immunology, immunogenomics, and computational modeling are highlighted as pathways toward predictive, decision-support-oriented frameworks. By positioning ADE platforms as immune assessment infrastructures rather than isolated assays, this review underscores their value for mechanistic inquiry, translational interpretation, and preparedness-oriented responses to emerging viral threats in the absence of definitive correlates of protection.

Pathogens doi: 10.3390/pathogens15050489

Authors: Chrispian Mamudi Purnamawati Panca Andana Prayudi Santoso Lidya Chaidir Arto Soeroto

Tuberculosis (TB) remains a leading cause of infectious mortality worldwide, reflecting persistent gaps in diagnosis, risk stratification, and treatment monitoring. Host RNA transcriptomic signatures have emerged as promising tools for capturing dynamic immune responses across the TB disease spectrum. Among these, the six-gene RISK6 signature has attracted attention due to its parsimonious design and potential for clinical translation. This review provides a clinically oriented synthesis of current evidence on host transcriptomic biomarkers, with a particular focus on the application of RISK6 in diagnosis, prediction of disease progression, and treatment monitoring. Available data suggest that RISK6 demonstrates promising but context-dependent diagnostic performance and represents a versatile host-response biomarker across multiple clinical applications. However, variability across populations and the limited evidence in multidrug-resistant TB remain important constraints. In practice, RISK6 is unlikely to function optimally as a standalone biomarker. Its clinical value appears greater when interpreted within integrated frameworks that combine transcriptomic, microbiological, and clinical data. Further validation in diverse populations and real-world settings will be essential to support meaningful clinical implementation.

Pathogens doi: 10.3390/pathogens15050488

Authors: Júlia dos Santos Fonseca Tábata Alves do Carmo Bianca de Oliveira Botelho Vital Thalita Suelen Avelar Monteiro Huarlen Marcio Balbino Huarrisson Azevedo Santos Vagner Tebaldi de Queiroz Fabio Ribeiro Braga Jackson Victor de Araújo

Although microbial biopesticides are expanding rapidly, transforming nematophagous fungi into consistent and shelf-stable products remains a challenge. A key limitation is that fungal propagules must remain viable throughout production, storage, and delivery to ensure their efficacy in the field. This review examines formulation strategies that improve the stability, deployment, and performance of fungal biocontrol agents against gastrointestinal helminths in domestic animals and plant-parasitic nematodes. In veterinary systems, predatory fungi such as Duddingtonia flagrans primarily target infective larvae after surviving gastrointestinal transit and germination in feces. In contrast, ovicidal fungi, including Pochonia chlamydosporia, Purpureocillium lilacinum, Trichoderma spp., and Mucor spp., primarily act against helminth eggs and coccidian oocysts. This functional complementarity highlights the potential of combined fungal formulations to improve their control efficacy. We also discuss the currently available D. flagrans-based commercial products, BioWorma® and Bioverm®, and the practical challenges associated with dosing, administration, and farm adoption. In agriculture, we show that the Brazilian market is dominated by solid fungal nematicides designed to reduce water activity and prolong shelf life, although liquid- and oil-based systems remain relevant for specific applications. Across both sectors, the review identified formulation design, rather than fungal species alone, as a critical determinant of product performance. Emerging advances, such as microencapsulation, UV-protective matrices, improved seed-coating biopolymers, nanobiotechnology, and fungal-derived bioactive products, indicate that future progress will depend on target-oriented formulations capable of increasing stability, controlled release, and resilience under environmentally variable conditions, including those imposed by climate change.

Pathogens doi: 10.3390/pathogens15050487

Authors: Andrea Nikčević Leona Radmanić Matotek Silva Šiftar Lorna Stemberger Marić Goran Tešović Snjezana Zidovec-Lepej

Background: Epstein–Barr virus (EBV)-associated infectious mononucleosis (IM) elicits a robust cellular immune response; however, systemic chemokine profiles in pediatric IM and their diagnostic relevance remain insufficiently characterized. This study evaluated proinflammatory chemokine expression in children with acute EBV-associated IM and its relationship with disease presence and severity. Methods: In this retrospective study, 64 children with confirmed acute EBV-associated IM and 16 healthy controls were included. Clinical severity was classified using the Severity of Mononucleosis (SOM) scale. Plasma concentrations of 12 chemokines were quantified by bead-based flow cytometry. Groups were compared using nonparametric tests, and logistic regression with cross-validation identified predictors distinguishing IM from controls. Results: CXCL9, CXCL10, and CXCL11 concentrations were significantly elevated in IM patients compared with controls across all severity strata (p ≤ 0.011), with no differences between SOM categories. CXCL5 concentrations were lower in severe (SOM ≥ 2) than moderate disease (p = 0.037). Other chemokines showed no significant differences. CXCL9 and CXCL10 effectively distinguished IM (AUC = 0.86, sensitivity = 0.71, specificity = 0.96). Conclusions: IFN-γ–inducible chemokines CXCL9–11 are markedly elevated in pediatric EBV-associated IM, irrespective of clinical severity, whereas CXCL5 may be associated with disease severity. Prospective validation of these preliminary findings is strongly warranted.

Pathogens doi: 10.3390/pathogens15050486

Authors: Ri De Yanpeng Xu Hanhaoyu Fu Liping Jia Linqing Zhao

Background: Four genotypes of human bocaviruses (HBoVs) have been identified, with only HBoV1 being detected in respiratory specimens, and with HBoV2 being the predominant human bocavirus in fecal specimens, which implies different tissue tropisms for HBoV1 and HBoV2. It is vital to determine the factors that influence the tissue tropisms. Methods: The major capsid proteins VP3 of HBoV1 and HBoV2 were expressed in eukaryotic cells. Then co-immunoprecipitation (Co-IP) and liquid chromatography–tandem mass spectrometry (LC-MS/MS) (IP-MS) was employed, along with Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses, to screen host proteins interacting with VP3 of different genotypes. Subsequently, in vitro pull-down assays were conducted to verify the direct virus–host interaction proteins with VP3. Furthermore, molecular docking was performed to predict the interaction interfaces between viral and host proteins. Results: Through IP-MS and enrichment analyses, 50 host proteins that displayed ≥10-fold differential binding affinities between HBoV1 VP3 and HBoV2 VP3 were identified. Among these, seven were considered as high-confidence candidate interactors. Notably, SPLUNC1 and VAMP8 showed predominant expression in respiratory and intestinal tissues, respectively. Subsequent in vitro pull-down assays confirmed that SPLUNC1 specifically bound to HBoV1 VP3, whereas VAMP8 specifically interacted with HBoV2 VP3. Molecular docking analysis further revealed that the binding between SPLUNC1 with HBoV1 VP3, as well as VAMP8 with HBoV2 VP3, was stabilized by extensive hydrophobic interfaces along with specific hydrogen bonds. Conclusions: The specific interactions of host proteins SPLUNC1 with HBoV1 VP3 and VAMP8 with HBoV2 VP3, respectively, provided fundamental evidence that the distinct tissue tropisms of HBoVs may be governed by specific host factors.

Pathogens doi: 10.3390/pathogens15050484

Authors: Aly Derbalah Ahmed Mohamed Nehad El-Gammal Warda Hussain Amany Hamza Ahmed Alhusays Ayman Omar Saleh Alhewairini

Chocolate spot, caused by the ascomycete fungus Botrytis fabae, is a devastating foliar disease and a major constraint on the quality and yield of faba beans (Vicia faba). Monoterpenes, such as carvone, cineole, and linalool, are often considered natural-identical alternatives to synthetic chemicals. Therefore, this study was carried out to assess the antifungal activity of some eco-friendly control agents (carvone, cineole, and linalool) against B. fabae, the causative agent of chocolate spot disease in faba beans, through growth inhibition assays in vitro. Furthermore, the efficacy of the tested monoterpenes for reducing the severity of chocolate spot disease in faba beans was evaluated under field conditions. Moreover, these eco-friendly control agents activate plant defense enzymes (phenylalanine ammonia-lyase, polyphenol oxidase, and peroxidase) as a self-defense mechanism against pathogen attacks of faba bean plants were investigated. Moreover, the impact of the tested monoterpenes on growth and yield characters of faba bean was evaluated. The results indicated a significant decrease in B. fabae growth following a treatment with the tested compounds compared to untreated controls. In field trials, treated faba bean plants exhibited a notable reduction in disease severity. Additionally, the application of monoterpenes enhanced the activity of defense enzymes (phenylalanine ammonia-lyase, polyphenol oxidase, and peroxidase), which are integral to plant defense mechanisms. Treatments also resulted in significant improvement growth and yield characters of faba bean. These findings suggest that the tested monoterpenes could serve as a control strategy for managing B. fabae, offering an environmentally sustainable alternative to conventional fungicides.

Pathogens doi: 10.3390/pathogens15050485

Authors: Theara Teng Sarin Neang Bruno M. Ghersi Cora Cunningham Daniel Nguyen Felicia B. Nutter Veasna Duong Thavry Hoem Sothyra Tum Theary Ren Dina Koeut Sam Eang Huon Sothealy Oeun Jonathon D. Gass Janetrix Hellen Amuguni Daniele Lantagne Tristan L. Burgess

In Cambodia, farmers construct artificial household bat roosts to collect and sell guano as fertilizer. We investigated farming practices and attendant spillover risks using (1) surveys on guano production; (2) an estimation of bat population size and species present using carcasses, visual identification, and audio recordings; (3) surveys of guano-producing and neighboring households on water, sanitation, and hygiene practices; and (4) the testing of guano and household food, water, and surfaces for coronaviruses using RT-qPCR. Bat roosts are constructed using dried palm leaves with coconut tree and/or steel/concrete supports. Roosting areas ranged from 42 to 327 m2, bat abundance varied from 0 to 11,187, guano production was between 5 and 120 kg/week, guano yields were from 0.15 to 0.4 kg/m2/week, and farmers earned USD ~100–200/household/month. Higher guano production in the peak (normally wet) season was associated with greater bat abundance (p = 0.016). The lesser Asiatic yellow house bat (Scotophilus kuhlii) was the only bat species identified. Roosts were <20 m from guano-producing households. Neighbors and households’ hygiene risks included not having handwashing stations and not covering food in storage/while drying. Coronaviruses (Alphacoronaviruses or Infectious Bronchitis Virus) were detected in 14.6%, 17.3%, 2.9%, 1.4%, and 0.0% of guano, urine, household surface, food, and water samples, respectively. While guano farming offers economic benefits, spillover risks exist. Safe guano collection and storage, handwashing, and food covering in guano-producing communities are necessary to mitigate spillover risks.

Pathogens doi: 10.3390/pathogens15050483

Authors: Ruihong Xiao Yanrui Bai Wenxiu Liu Hui Sun

Fusobacterium nucleatum (Fn) has emerged as one of the most extensively studied tumor-associated opportunistic pathogens in colorectal cancer (CRC). The central question in Fn–CRC research has shifted from species-level detection or enrichment toward identifying specific lineages with enhanced persistence and tumor-promoting potential under defined host and ecological contexts. Accumulating evidence suggests substantial heterogeneity within Fn at the subspecies and clade levels. Among these, the F. nucleatum subsp. animalis C2 (Fna C2) lineage has been proposed as a candidate high-risk clade with potentially greater adaptability to the gastrointestinal tract and tumor microenvironment. However, current support for Fna C2 is derived mainly from ecological enrichment, comparative genomics, inferred metabolic features, and limited functional observations, while direct clinical and mechanistic validation at the clade level remains limited. Fn has been implicated in CRC progression through multiple interconnected processes, including adhesion and colonization, host signaling activation, inflammatory amplification, immune suppression, and metabolic adaptation. Notably, these pathogenic outputs are unlikely to be uniformly distributed across all Fn lineages, but instead appear to be shaped by the combined influence of bacterial lineage, host molecular context, microbial community structure, and spatial organization within the tumor microenvironment. In this review, we summarize the lineage heterogeneity of Fn, its association with CRC, and the underlying host–pathogen interaction mechanisms. We further discuss implications for high-resolution stratification, risk classification, and clinical translation, emphasizing the need to move from species-level associations toward lineage-resolved and context-aware frameworks.

Pathogens doi: 10.3390/pathogens15050482

Authors: Olivia Alicia Reynoso-Ducoing Elsa Gabriela Díaz-Ramírez Elia Torres-Gutiérrez Mauro Omar Vences-Blanco Berenice González-Rete Yolanda Guevara-Gómez Margarita Cabrera-Bravo Paz María Silvia Salazar-Schettino

Food deprivation induces intestinal adaptations in Triatoma pallidipennis, a hematophagous insect with intermittent feeding habits. The ability to survive long periods without food promotes the persistence of this vector in the environment and contributes to its evolutionary success. This study employed one- and two-dimensional electrophoretic techniques combined with Western blot to evaluate the abundance of α-tubulin, β-actin, and the heat shock protein HSP70. These proteins were more abundant in the anterior midgut tissue of unfed insects than in that of fed insects. As these responses were similar in females and males, the observed adaptations primarily depend on feeding status and intestinal region. These findings provide further insight into the intestinal physiology of T. pallidipennis, a vector of the flagellate parasite Trypanosoma cruzi, the causative agent of Chagas disease.

Pathogens doi: 10.3390/pathogens15050481

Authors: Ali Wahdan Mahmoud Ezzat Amal Emam Walaa A. Husseiny Mohamed Abou El-Atta Ehab M. Abd-Allah Ahmed M. A. Meligy Sherief M. Abdel-Raheem Faisal Almathen Salah Al-Shami Saad I. Al-Sultan Ahmed Alfifi Wael El-Deeb Marwa E. Abo Hashem

This notice provides additional clarification to the original publication [...]

Pathogens doi: 10.3390/pathogens15050480

Authors: Min A Lim Na Young Hong Sook Hee Park Myung Hee Kim Youkyoung Kim Ji Hong Park Dong Gyu Park Hee Young Hwang Seok Ju Yoo Ji Hyuk Park Kwan Lee

Postoperative endophthalmitis is a rare but severe complication of cataract surgery that may lead to significant visual impairment and can occasionally present as clusters or outbreaks linked to lapses in infection control. On 29 October 2025, three cases of postoperative endophthalmitis following cataract surgery at an ophthalmology clinic (Clinic A) in Yeongju, Republic of Korea, were reported to public health authorities. All cases were confirmed as Pseudomonas aeruginosa (P. aeruginosa). An epidemiological investigation was conducted to identify the source and transmission route; all 54 patients who underwent cataract surgery at Clinic A in October 2025 (62 procedures, including eight bilateral cases) were included. Infection control practices were assessed through on-site inspection, staff interviews, medical record review, and telephone surveys. Environmental samples were collected and cultured selectively for P. aeruginosa. Isolates were analyzed using pulsed-field gel electrophoresis (PFGE) to assess genetic relatedness. Among 54 surgical patients, three developed endophthalmitis (attack rate: 5.6%). All cases occurred in patients operated on 23 October by the same surgeon; no additional cases were identified. P. aeruginosa was isolated from all three patients. Of 45 environmental samples, three were positive: the laundry room drain, the sink drain in the local anesthesia room, and the interior of cassette No. 2 and instruments within the operating room steam sterilizer. PFGE showed 95.7% band pattern similarity between patient isolates and those from the sterilizer. This outbreak was attributed to P. aeruginosa, with the steam sterilizer identified as the most probable source. Inadequate infection control and sterilization practices likely contributed. These findings highlight the critical importance of strict sterilization monitoring and adherence to infection prevention and control practices to prevent recurrence.

Pathogens doi: 10.3390/pathogens15050479

Authors: Watcharapong Mitsuwan Juthatip Jeenkeawpieam Ratchadaporn Boripun Noppharat Tanthanathipchai Ozioma Forstinus Nwabor Phirabhat Saengsawang

Otitis externa in dogs is primarily caused by Malassezia pachydermatis. Treatment involves antifungal and antiseptic agents; however, resistance among causative organisms has been noted. Pomelo (Citrus maxima) is a source of bioactive compounds with antimicrobial activity. Its extract mainly includes essential oils, which are mostly applied for alternative treatment for M. pachydermatis. The study aimed to investigate the anti-M. pachydermatis effects of pomelo peel extracts and their potential use in topical solutions for canine infections. M. pachydermatis was isolated from dogs and confirmed with Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF/MS). Antifungal susceptibility of M. pachydermatis to itraconazole was evaluated. Phytochemicals of essential oil and crude extract from C. maxima peel were determined using Gas Chromatograph–Mass Spectrometry (GC-MS/MS). In addition, the antifungal activity of the extracts was assessed using an agar plate dilution assay. The essential oil was formulated into a prototypic topical solution, and its effects on M. pachydermatis were observed in vitro. The prevalence of M. pachydermatis was 42%, with 53% having yeast on both ear sides. The minimum inhibitory concentrations (MIC) of itraconazole, essential oil, and crude extract to M. pachydermatis were 0.03–0.25 µg/mL, 1.0% v/v, and >200 mg/mL, respectively. The prominent phytochemicals in peel extracts were meranzin hydrate and D-limonene, identified in the crude extract and essential oil, respectively. Moreover, a topical solution containing essential oils inhibited M. pachydermatis growth and showed destructive effects on the yeast cell wall at higher concentrations. The essential oil exhibits antifungal activity against M. pachydermatis, primarily due to the high concentration of D-limonene. The growth was inhibited completely at MIC, observed over a 5-day period. Furthermore, the prototypic topical solution demonstrated an anti-M. pachydermatis effect. These findings suggest potential veterinary applications for pomelo peel extract, though further studies are necessary to assess stability, mechanism of action, and industrial suitability.

Pathogens doi: 10.3390/pathogens15050478

Authors: Robin C. Anderson Delila D. Dominguez Megan R. Shaw Casey N. Johnson Samat Amat Jackie M. Kotzur Merritt L. Drewery Patricia J. Baynham Ken J. Genovese Tawni L. Crippen Ryan J. Arsenault

Salmonella in gut habitats have traditionally been thought to conserve energy for growth via fermentation. However, recent reports indicate that ingested Salmonella can stimulate host-derived nitrate accumulation in the mucosal microenvironment, thereby enabling growth through nitrate respiration. Sodium tungstate is an effective treatment that inhibits the growth of certain nitrate-respiring bacteria, including Escherichia coli, Paracoccus and Proteus, when cultured under gut simulating conditions or within the gut of experimentally treated mice. This inhibitory effect is hypothesized to occur by inactivation of molybdenum-containing enzymes required for nitrate metabolism. Information is lacking on whether tungstate can inhibit the growth of Salmonella, particularly in the presence of culturable gut microbiota. Therefore, the objectives of this study were to evaluate the effects of sodium tungstate on Salmonella during pure culture or when cultured with freshly collected bovine rumen microbiota and to assess its impact on fermentation as well as nitrate and nitrite metabolism within the rumen microbial cultures. Our results indicate that 50 mM sodium tungstate treatment, whether alone or in combination with 5 mM nitrate, markedly increased the growth of Salmonella serovars Newport, Dublin and Typhimurium during pure culture. Moreover, during in vitro incubation, increased growth of experimentally inoculated S. Newport as well as wildtype E. coli and lactic acid bacteria was observed with ruminal microbiota treated with 100 mM tungstate when compared to non-tungstate-treated controls. Effects of tungstate on nitrate and nitrite metabolism were as expected during pure and mixed culture. When cultured with reduced tungsten rather than tungstate, the latter being bound to four oxygen atoms, an inhibitory effect on the growth of S. Newport was observed and effects on nitrate and nitrite metabolism were consistent with those observed with tungstate. These results suggest that, under conditions used in the present experiments, tungstate may have served as a source of oxygen for respiration above that achieved with nitrate alone. While this hypothesis has yet to be proven, it is supported by an adverse effect of tungstate, whether alone or in combination with 5 mM nitrate, on methane and volatile fatty acid production by the ruminal microbiota when compared to untreated or nitrate-only-treated microbiota.

Pathogens doi: 10.3390/pathogens15050477

Authors: Swamy R. Adapa Faiza A. Siddiqui Rays H. Y. Jiang

Heme metabolism is central to the biology of malaria parasites and to the mechanism of action of artemisinin-based therapies. Within malaria-infected red blood cells (RBCs), heme-related chemistry arises from multiple nested metabolic sources that function as “Russian dolls”: the truncated heme biosynthetic capacity of the host erythrocyte, the parasite’s own heme synthesis pathway, and host heme released through hemoglobin digestion in the parasite food vacuole. These overlapping metabolic layers create distinct pools of heme that can influence redox balance and drug activation. Recent studies highlight that exogenous 5-aminolevulinic acid (5-ALA) can perturb host heme biosynthesis in infected erythrocytes, potentially increasing intracellular levels of the heme intermediate protoporphyrin IX and sensitizing parasites to oxidative stress. However, the extent to which such metabolic perturbations affect artemisinin susceptibility depends strongly on parasite stage and exposure duration. Here we review the compartmentalized architecture of heme metabolism in malaria-infected RBCs and discuss how these nested vulnerabilities may be exploited for therapeutic intervention.

Pathogens doi: 10.3390/pathogens15050476

Authors: Jingrui Zhang Xiao Liu Li Liu Yeshun Fan Zhiwen Sun Mengjie Li Yanwen Xiong Zengbin Liu Yanfang Li Lili Yu Hongru Feng Duochun Wang Jingxiao Zhang

Patients undergoing cervical conization for cervical intraepithelial neoplasia grade II (CIN II) may develop postoperative bloodstream infections (BSIs), and multidrug-resistant (MDR) Escherichia coli isolates with distinct resistance profiles can complicate antimicrobial management. In this case-based study, two E. coli strains, HBFY-1 and HBFY-2, were recovered from blood cultures obtained from a 38-year-old CIN II patient with postoperative fever. The isolates were characterized by antimicrobial susceptibility testing, whole-genome sequencing, conjugation assays, and comparative genomics against publicly available genomes matched by sequence type and serotype. Fever occurred on postoperative day 2. HBFY-1 was fluoroquinolone-resistant; belonged to ST744/O101:H9; carried the extended-spectrum β-lactamase (ESBL) gene blaCTX-M-27; was phenotypically confirmed as an ESBL producer; and grouped within a multi-source near-neighbor clade consistent with a conserved fluoroquinolone-associated resistance backbone in ST744/O101:H9. HBFY-2 was carbapenem-resistant; belonged to ST48/O113:H32; carried blaNDM-5 on an IncY-associated plasmid bin; was phenotypically confirmed as a metallo-carbapenemase producer; and did not harbor any ESBL gene. Within the matched ST48/O113:H32 dataset, blaNDM-5 was detected only in HBFY-2, which clustered within an Asia-enriched lineage, including China-derived human and swine genomes. The blaCTX-M-27-associated and blaNDM-5-associated elements were transferred to E. coli C600 at frequencies of 5.3 × 10−2 and 4.6 × 10−6, respectively, and transfer of the blaNDM-5-associated element imposed no detectable growth penalty under the tested conditions. As this study is based on a single clinical case, the findings should be interpreted cautiously, yet they still highlight the potential value of integrating susceptibility testing with rapid genomic characterization for identifying mobilizable carbapenem-resistance platforms.

Pathogens doi: 10.3390/pathogens15050475

Authors: Michele Macrelli Martina Ossola Giovanni Sala Damiano Accurso Monica Caffara Andrea Gustinelli Marco Farioli Cristian Salogni

The increasing consumption of fish has raised public health concerns regarding fish-borne zoonotic helminths (FBZHs), which are recognized as significant food-borne parasites worldwide. In freshwater environments, Clinostomum complanatum, Opisthorchis felineus, Pseudamphistomum truncatum, Dibothriocephalus latus and Eustrongylides excisus are of particular concern in Italy and neighbouring countries. This study aimed to assess the prevalence of these FBZHs in five commercially and ecologically relevant freshwater fish species from Lake Varese, a heavily anthropized and understudied basin in northern Italy. A total of 59 fish were examined via necropsy and stereomicroscopic inspection of skeletal muscles. Only Eustrongylides spp. larvae were detected, with a prevalence of 16.9%. Molecular analysis (ITS region) identified them as E. excisus. This study reports, for the first time in Western Europe, E. excisus in Sander lucioperca (p = 12.5%) and Esox lucius (p = 8.3%). The highest prevalence occurred in Silurus glanis (p = 37.5%), followed by Perca fluviatilis (p = 25.0%), while Tinca tinca showed no infection. These findings confirm that among the FBZHs considered, E. excisus is currently present and expanding both in host range and geography in Italian lakes, underscoring the need for updated epidemiological data to support risk assessment, food safety and zoonotic parasite control in freshwater fisheries.

Pathogens doi: 10.3390/pathogens15050474

Authors: Irene Scarvaglieri Maria Antonia De Francesco Maria Alberti Federico Cesanelli Martina Salvi Giorgio Tiecco Francesco Castelli Eugenia Quiros-Roldan

Background: Influenza A(H3N2) viruses remain a major public health concern due to their rapid antigenic evolution and association with severe disease, particularly among high-risk populations. During the 2025–2026 influenza season, a marked epidemiological shift was observed in Europe, with the emergence and predominance of the A(H3N2) subclade K (J.2.4.1). Objectives: This narrative review aims to provide an integrated overview of the epidemiology, evolutionary dynamics, and public health implications of subclade K, with a particular focus on its impact on vaccine effectiveness, in comparison with the 2024–2025 influenza season. Methods: A non-systematic literature review was conducted using major scientific databases and official public health sources, including WHO and ECDC reports. Recent surveillance data, genomic analyses, and epidemiological updates were included. Given the rapidly evolving evidence base, selected preprint studies were also considered and interpreted with caution. Results: The 2025–2026 influenza season in Europe was characterized by a relative genetic convergence, with subclade K accounting for the majority of A(H3N2) sequences. This variant demonstrated a clear selective advantage and was associated with an earlier and more intense epidemic peak. Molecular analyses indicate the accumulation of multiple mutations in the hemagglutinin protein, particularly within key antigenic sites, contributing to immune escape. These evolutionary changes have important implications for vaccine effectiveness, with current estimates suggesting moderate protection against infection but preserved effectiveness against severe outcomes. Antigenic mismatch, manufacturing constraints, and host-related factors further contribute to reduced vaccine performance. Conclusions: The emergence and rapid spread of subclade K highlight the dynamic nature of influenza virus evolution and its impact on public health. Continuous genomic surveillance and timely vaccine updates remain essential. Despite suboptimal effectiveness against infection, influenza vaccination continues to provide significant protection against severe disease and should remain a cornerstone of prevention strategies.

Pathogens doi: 10.3390/pathogens15050473

Authors: Donghui Liu Heng Quan Mengyao Liu Lingling Xiao Lei Jiao Xiaowei Gong Qiaoying Zeng Qiwei Chen

Background: The intracellular pathogen Brucella requires biotin for survival, yet the role of its de novo synthesis intermediate enzyme, BioA, in virulence remains undefined. This study investigates the contribution of BioA to the pathogenicity of Brucella abortus. Methods: We constructed a ΔBioA mutant in Brucella abortus 104M via homologous recombination and characterized its phenotype using growth assays, electron microscopy, macrophage infection models, and murine splenic colonization. Virulence gene expression was quantified by RT-qPCR. Results: The ΔBioA mutant exhibited severe growth auxotrophy in a biotin-deficient medium and displayed damaged outer membrane integrity. Furthermore, intracellular survival in macrophages was reduced by approximately 95% compared to the wild-type strain at 48 h post-infection. Notably, mice infected with the mutant showed a significant decrease in both splenic bacterial loads and spleen weight at 3 weeks, concomitant with a marked downregulation of VirB type IV secretion system (T4SS) genes. Conclusions: This study is the first to identify BioA as a critical nexus linking biotin metabolism to Brucella virulence. We demonstrate that BioA deficiency attenuates pathogenicity by impairing both structural integrity and the transcription of key virulence-related genes (VirB operon), thereby nominating BioA as a novel and promising target for anti-brucellosis interventions.

Pathogens doi: 10.3390/pathogens15050472

Authors: Basant Abdulrahman Shabboo Rahimi Aqdam Matteo Mosca Hanaa Ahmed-Hassan Melissa Razcon-Echeagaray Lia Popa Sabine Gilch Baaissa Babelhadj Gabriele Vaccari Hermann M. Schätzl

Prion diseases are fatal neurodegenerative disorders that affect humans and animals, caused by the conformational conversion of the normal cellular prion protein (PrPC) into its misfolded, infectious isoform PrPSc. Recently, camel prion disease (CPrD) was identified in dromedary camels (Camelus dromedarius) in Algeria. Due to the potential implications for animal and human health, as well as the possible socio-economic impact in Mediterranean regions where camels play a pivotal role as a source of food, in-depth characterization of camel prions is important to increase our understanding of camel prion disease. We developed a neuronal cell line model for studying the molecular features of camel prion infection. We genetically edited mouse neuronal CAD5 cells to generate CAD5 PrP knockout (KO) cells. We then used lentiviral transduction to generate CAD5 cells expressing camel PrP (CAD5-camel-PrP). Following infection of these cells with a CPrD-positive camel brain homogenate, we observed PrPSc signals at various passages, as indicated by immunoblotting analysis. RT-QuIC (Real-Time Quaking-Induced Conversion) assays further supported these findings, demonstrating transient prion conversion activity in the CPrD-infected CAD5-camel-PrP cells. Taken together, our data describe the first neuronal cell line permissive to camel prion infection, a novel in vitro tool for mechanistic studies of camel prion disease.

Pathogens doi: 10.3390/pathogens15050471

Authors: Mehmet Ozturk Huseyin Cihan Demirel Ilker Seckiner Taner Haciosmanoglu Muharrem Baturu Semih Turk Onur Zeytun Kaya Horasanli

Introduction: Postoperative infectious complications following retrograde intrarenal surgery (RIRS) remain a significant clinical challenge due to their potential progression to sepsis. This study aimed to identify perioperative risk factors associated with infection and to develop a practical risk stratification model. Materials and Methods: A total of 1949 patients who underwent RIRS in two centers between 2014 and 2025 were retrospectively analyzed. Patients were grouped according to irrigation method, and infectious outcomes including febrile urinary tract infection (UTI), sepsis, and septic shock were evaluated. Multivariable logistic regression analysis identified independent predictors of postoperative infection. Results: Overall, infectious complications occurred in 158 patients (8.1%), with no significant difference in total infection rates between the two groups. In the overall cohort, older age (OR 1.01; p = 0.045), preoperative JJ stenting (OR 1.48; p = 0.038), longer operative time (OR 1.01; p = 0.049), and a history of preoperative UTI requiring antibacterial treatment (OR 68.45; p < 0.001) were independent predictors of postoperative infection. These variables informed the development of the RIRS-STAMP score; the final combined model showed good discrimination (AUC 0.84, 95% CI 0.80–0.88) and was internally validated using 1000 bootstrap resamples. Discussion: These findings highlight the multifactorial nature of infection risk after RIRS and emphasize the importance of both host factors and procedural dynamics in infection development. The RIRS-STAMP score was developed based on these findings. Conclusions: The model can enable early identification of high-risk patients and supports individualized perioperative management; however, prospective external validation is required before routine clinical use.

Pathogens doi: 10.3390/pathogens15050470

Authors: Polly Roy

Bluetongue virus (BTV), the prototype of the genus Orbivirus, infects livestock, causing high morbidity and mortality and impacting global trade. BTV is a non-enveloped, double-capsid virus, composed of seven structural proteins and a genome of 10 double-stranded RNA segments. This manuscript highlights our group’s recent findings on the molecular and structural mechanisms underlying BTV entry and assembly during replication. Viral entry is a stepwise, pH-dependent process. The outermost protein, VP2, attaches to sialic acids and senses the acidic pH of early endosomes, triggering their dissociation. Subsequently, the second outer capsid protein, VP5, undergoes major changes in late endosomes, forming a membrane-penetrating pore that releases the transcriptionally active inner core into the host cytoplasm. Core assembly also proceeds stepwise and requires the accurate packaging of 10 positive-sense RNA segments. These segments form an RNA–RNA interaction network independent of viral proteins, beginning with the smaller segments and guiding the complete genome assortment. The small capsid protein, VP6, interacts with VP3 to facilitate RNA encapsidation. While infectious cores assemble in vitro without non-structural proteins, NS2 is essential for the in vivo formation of viral inclusion bodies via liquid–liquid phase separation, concentrating viral components and promoting genome assembly. These comprehensive characterizations of BTV provide a foundation for future control strategies against related reoviruses.

Pathogens doi: 10.3390/pathogens15050469

Authors: Elena-Teona Coșovanu Teodora Ana Balan Eric-Oliviu Coșovanu Silvia Ionescu Costin Damian Antoneta Dacia Petroaie Elena-Adorata Coman Mihaela Grigore Demetra Socolov Raluca Anca Balan Luminita Smaranda Iancu Irina Draga Căruntu Ramona Gabriela Ursu

Neonatal infections remain a leading cause of morbidity and mortality worldwide, particularly among preterm and low-birth-weight infants and in low- and middle-income countries. This burden has intensified with the global increase in multidrug-resistant (MDR) bacteria, especially in neonatal intensive care units, where prolonged hospitalization, invasive interventions, and exposure to broad-spectrum antibiotics promote colonization, transmission, and invasive infection. In this narrative review, we explore the epidemiology and microbiological characteristics of MDR bacterial infections in newborns, alongside their associated risk factors, diagnostic challenges, treatment outcomes, and prevention strategies. Across different settings, Gram-negative pathogens, particularly Klebsiella pneumoniae, Escherichia coli, and Acinetobacter baumannii, account for a substantial proportion of severe neonatal infections, whereas methicillin-resistant Staphylococcus aureus remains important in selected units. The risk of MDR infection is driven by a complex interplay of factors, ranging from maternal and perinatal exposures to the inherent immunological vulnerability of newborns, hospital-based transmission, antibiotic selection pressure, and structural deficiencies in healthcare infrastructure. Diagnosis remains challenging because clinical presentations are nonspecific and culture-based methods are constrained by low blood volumes, prior antimicrobial exposure, and delayed turnaround times. Treatment is increasingly complicated due to resistance to standard empirical regimens, substantial regional variation in susceptibility profiles, and limited neonatal pharmacokinetic and safety data for reserve agents. Current evidence mainly supports surveillance-informed empirical therapy, susceptibility-guided treatment adjustment, antimicrobial stewardship, and strict infection prevention measures. Future progress will require neonatal-specific clinical trials, harmonized surveillance systems, stronger molecular epidemiology, and more equitable access to microbiological diagnostics and effective treatment.

Pathogens doi: 10.3390/pathogens15050468

Authors: Maria Monica Florina Moraru Ana-Maria Marin Dan-Cornel Popovici Azzurra Santoro Federica Santolamazza Radu Blaga Kalman Imre Narcisa Mederle

The Eurasian lynx (Lynx lynx) is an apex predator and an important sentinel for trophically transmitted helminths acquired via predation on wild ungulates. On 2 March 2022, an adult male lynx that was road-killed in the Apuseni Mountains (Surducel hunting ground, Bihor County) was collected, frozen for biosafety, and a necropsy was performed. Taeniid cestodes were detected, with a total intestinal burden of nine adult specimens. Genetic analyses confirmed Taenia lynciscapreoli, and the obtained sequences were deposited in GenBank (PV843597, PV855065, PV844409). Phylogenetic inference based on cox1 assigned the Romanian isolate within the European cluster, distinct from the Chinese isolate, while showing genetic proximity to Taenia sp. (MW846305) that have been reported from a lynx in China. This study represents the first molecular identification of T. lynciscapreoli in the Eurasian lynx in Romania and, to our knowledge, the first record from Southeastern Europe.

Pathogens doi: 10.3390/pathogens15050467

Authors: Despoina Kypraiou Angelos Sourris Eirini Astrinaki Efsevia Vitsaxaki Stamatina Saplamidou Maria Vakonaki Kyriaki Tryfinopoulou Georgios Chamilos Petros Ioannou Diamantis Kofteridis

Background: Vancomycin-resistant Enterococcus (VRE) bacteremia represents a major therapeutic and epidemiological challenge, particularly in regions with high antimicrobial resistance rates such as Southern Europe. Longitudinal local data are essential to guide infection control and antimicrobial stewardship strategies. This study aimed to evaluate temporal trends in incidence, management, and mortality of VRE bacteremia in a tertiary care center in Greece over a six-year period, including comparison before and after the coronavirus disease 2019 (COVID-19) pandemic. Methods: This retrospective observational study included adult patients with VRE bacteremia at the University Hospital of Heraklion, Greece, from 2018 to 2023. Demographic and clinical data, such as the Pitt Bacteremia Index (PBI), as well as microbiological, and treatment data were collected from patient records. Incidence was calculated per 10,000 patient-days. Comparisons were performed between survivors and non-survivors and between pre- and post-COVID-19 eras. Multivariate regression analysis was used to identify predictors of in-hospital mortality. Results: A total of 96 patients were included (mean age 68.6 ± 14.5 years; 56.3% male). The incidence of VRE bacteremia increased more than five-fold during the study period, from 0.242 cases per 10,000 patient-days in 2018 to a peak of 1.344 per 10,000 patient-days in 2022, remaining elevated in 2023 (1.001 per 10,000 patient-days). The overall in-hospital mortality was 54.2%. Non-survivors had significantly higher PBI scores compared to survivors (median 2.5 vs. 0, p = 0.005). In the multivariate analysis, higher PBI was independently associated with in-hospital mortality [odds ratio: 1.449 (95% confidence intervals: 1.166–1.801)]. Appropriate empirical therapy was administered in 41.7% of cases and was not significantly associated with survival. Post-COVID-19 patients were older (69.9 vs. 61.4 years, p = 0.0365), and antimicrobial regimens were more frequently adjusted according to susceptibility testing (55.7% vs. 18.2%, p = 0.0141), but mortality did not significantly differ between periods. Conclusion: VRE bacteremia incidence increased dramatically over the six-year study period in our tertiary center, with persistently high mortality exceeding 50%. Severity of illness at the diagnosis of bacteremia, as measured by the PBI, was an independent predictor of in-hospital mortality. Strengthened infection prevention measures, optimized antimicrobial stewardship, and early aggressive management are urgently needed to mitigate the growing burden of VRE bacteremia.

Pathogens doi: 10.3390/pathogens15050466

Authors: Dinara Imanbayeva Moh A. Alkhamis John M. Humphreys Andres M. Perez

Peste des Petits Ruminants (PPR) is a highly contagious viral disease of small ruminants and remains a major threat to food security and rural livelihoods across Africa, the Middle East, and Asia. In the Mediterranean, uneven outbreak reporting and intense spatial clustering hinder the identification of regions where environmental and anthropogenic conditions favor disease occurrence. This study applied an interpretable machine-learning ecological niche modeling framework to characterize PPR spatial suitability in North Africa. A merged outbreak dataset (n = 744) was compiled from the Food and Agriculture Organization (FAO) EMPRES-i and the World Animal Health Information System (WAHIS) databases for 2005–2026. Outbreak locations were linked to environmental and anthropogenic predictors, spatially thinned, and paired with randomly sampled pseudo-absences at a 1:1 ratio. After correlation-based screening and Boruta feature selection, four classifiers were compared under five-fold spatial block cross-validation: a generalized linear model (GLM), a support vector machine (SVM), Random Forest (RF), and extreme gradient boosting (XGBoost). All models showed good discriminatory performance. Random Forest (RF) and extreme gradient boosting (XGBoost) yielded the highest area under the receiver operating characteristic curve value (AUC = 0.94). Random Forest achieved the highest specificity, XGBoost achieved the highest sensitivity, and the support vector machine showed the most even sensitivity–specificity tradeoff among the machine-learning classifiers. Sheep density, mean diurnal temperature range, temperature seasonality, and human population density were consistently the dominant drivers. Predicted PPR suitability based on reported outbreaks was concentrated along the North African coastal belt and low across most arid inland regions. These findings suggest that passive surveillance is likely to be most informative in coastal production systems where host density, environmental suitability, and reporting opportunity overlap. At the same time, areas of lower reported-outbreak suitability should not be interpreted as disease-free and may require complementary active surveillance approaches.

Pathogens doi: 10.3390/pathogens15050465

Authors: Manuela Arbune Roxana-Elena Bogdan-Goroftei Alina-Viorica Iancu Diana-Sabina Radaschin Florin-Ciprian Bujoreanu Alin-Laurentiu Tatu Claudia-Simona Stefan

Syphilis and HIV are sexually transmitted disease (STDs) that interact synergistically. However, data on HIV–syphilis co-infection in Romania remain limited. We conducted a retrospective cohort study at a single Romanian HIV/AIDS Day Clinic, including 439 adult people living with HIV (PLWH) monitored between 2020 and 2025. Demographic, epidemiological, clinical, and laboratory data were collected, including HIV staging and syphilis history. Syphilis co-infection was identified in 81 patients (18.5%), and 61.5% met criteria for AIDS. Viral suppression was achieved in 82.2%, and 78.4% achieved CD4 counts >350 cells/mm3. Male sex, urban residence, unmarried status, sexual HIV transmission, genital condyloma, and other STIs were independently associated with syphilis. First episodes of syphilis were predominantly secondary (61%), neurosyphilis was present in 5%, and serofast evolution occurred in 12%, more frequently after reinfection. Among deceased patients, 20.9% had a history of syphilis, but co-infection was not significantly associated with mortality. Nine of 28 patients lost to follow-up had prior syphilis, suggesting a potential impact on retention in care. These findings indicate that HIV–syphilis co-infection is increasingly prevalent in Romania, driven primarily by behavioral factors, and highlight the need for targeted STD screening and prevention strategies among high-risk PLWH.

Pathogens doi: 10.3390/pathogens15050464

Authors: Meng Zhou Kun Ren Huan Mei Hang Yang Dongmei Li Weida Liu Lulu Zhang Xiaodong She

Vulvovaginal candidiasis (VVC) is a common gynecological infection, with Pichia kudriavzevii emerging as a significant pathogen due to its intrinsic fluconazole resistance and biofilm-forming capacity. This study investigates the antifungal efficacy and mechanisms of tannic acid (TA) against P. kudriavzevii, as well as its potential to reverse azole resistance across multiple Candida species with distinct resistance profiles. TA significantly inhibited P. kudriavzevii growth, surface colonization, and virulence gene expression at 3 μg/mL. Mechanistically, TA protected the human vaginal epithelial cell line VK2/E6E7 by reducing ROS levels, restoring mitochondrial membrane potential, and suppressing IL-1β and IL-18 release through modulation of the NLRP3-Caspase1-ASC axis. Furthermore, TA demonstrated synergistic activity when combined with azoles against five clinically azole-resistant Candida isolates spanning three Candida species with distinct resistance mechanisms: P. kudriavzevii (intrinsic), C. albicans (acquired), and N. glabrata (FKS-mediated). This study highlights TA as a promising natural therapeutic agent for P. kudriavzevii infections and offers a novel strategy for combating multidrug-resistant Candida through combination therapy.

Pathogens doi: 10.3390/pathogens15050463

Authors: Qiuchi Lv Lexi Li Ruifei Wang Shuaibing Han Hongwei Zhao Zhengde Xie Qiang He Chang Liu Lili Xu

Human adenovirus type 7 (HAdV-7) is a significant pathogen responsible for viral community-acquired pneumonia in children. To date, no specific antiviral agents have been approved for clinical use against HAdV infections. Traditional Chinese medicine (TCM) mixtures have shown promising potential in managing viral pneumonia. This study aimed to evaluate the antiviral activity of Yuanzhixingrenheji (YZ), a hospital-prepared TCM formulation from Beijing Children’s Hospital, against HAdV-7. Initial screening of four hospital formulations (Feiyanheji, Qingjieheji, Yindaizhikeheji, and Yuanzhixingrenheji) using a CCK-8 assay revealed that YZ exhibited the lowest cytotoxicity. In vitro, YZ pretreatment and post-infection treatment exhibited dose-dependent antiviral activity against HAdV-7 in A549 cells, significantly suppressing the DBP mRNA level and protein expression while reducing viral genome copies, HAdV-7-GFP fluorescence, hexon fluorescence, and DBP nuclear localization. In the hDSG2+/+ C57BL/6 mouse model of HAdV-7 infection, YZ effectively mitigated infection-induced body weight loss and substantially reduced viral loads in lung tissue. Furthermore, a clinical retrospective analysis indicated that YZ treatment significantly decreased post-hospitalization serum C-reactive protein levels of pediatric patients with HAdV infection in various disease severities. Compared with conventional treatment, YZ treatment also significantly reduced peak temperature and shortened the duration of fever in children with HAdV infection, supporting its therapeutic potential. In summary, this study provides the first integrated evidence from in vitro, in vivo, and clinical retrospective investigations, demonstrating that the TCM mixture YZ has significant anti-HAdV-7 activity and clinical efficacy. Characterized by a favorable safety profile and low economic burden, YZ is a promising candidate for the treatment of pediatric adenovirus pneumonia.

Pathogens doi: 10.3390/pathogens15050462

Authors: Edwin Omar Desales-Decaro Graciela Castro-Escarpulli Andres Saldaña-Padilla Alejandro Cravioto Hugo G. Castelán-Sánchez Armando Navarro-Ocaña

Shigella spp., and Escherichia coli exhibit notable genomic and phenotypic similarities, including serologically and genetically related somatic antigens. For example, the relationship among pathogenic strains E. coli 64474, O179, O188, and S. boydii O16 suggests a shared clonal origin. To evaluate their genomic proximity, a comparative genomics study was conducted using whole-genome sequencing. Comparative genomics involved rfb gene cluster regions and whole-genome comparisons. Phylogenomic inferences were performed using the virtual genome fingerprint (VGF) method with bootstrap support. The results revealed a high degree of genomic similarity and a close evolutionary relationship among E. coli strains, which also demonstrated genetic associations with clinically relevant pathotypes through the presence of virulence genes. Furthermore, serogroups 64474, O188, and S. boydii O16 exhibited close genetic relationships, suggesting that serotype 64474 could represent a novel serogroup, although its similarity to O188 indicates the influence of divergent factors. These findings support the hypothesis that these E. coli strains originated from a common clonal lineage, enhancing our understanding of serogroup diversity and the evolutionary dynamics within enteric pathogens.

Pathogens doi: 10.3390/pathogens15050461

Authors: Paloma Camacho-Aguilar Javier Alejandro Delgado-Nungaray Eire Reynaga-Delgado Orfil Gonzalez-Reynoso Libia Zulema Rodriguez-Anaya Luis Alfonso Muñoz Miranda Gabriel Rincón Enríquez Inocencio Higuera-Ciapara Luis Joel Figueroa-Yáñez

Helicobacter pylori (H. pylori) is a highly adaptable gastric pathogen with marked genomic plasticity. Whilst functional CRISPR-Cas systems provide adaptive immunity in many bacteria, they have not been identified in H. pylori, unlike CRISPR-like sequences. In this study, eight H. pylori genomes were analysed using the bioinformatics tools CRISPRCasFinder, CRISPRCasTyper, and CRISPRloci. A total of 25 CRISPR-like arrays were identified, showing high conservation (88%) both between and within strains, suggesting that these arrays are not random remnants but rather organised structures possibly involved in cellular processes. Notably, a structural association was observed between the CRISPR-like sequences and the cag pathogenicity island (CagA-PAI). Conversely, CagA-PAI instability in specific strains was observed in the presence of the TnpA and TnpB transposons. Furthermore, in strain 29CaP, CRISPR-like assemblies were located in genomic proximity to the prophage Helico 1961P, leading to the hypothesis of a compensatory or regulatory effect in the absence of CagA-PAI. Taken together, these findings indicate that CRISPR-like arrays in H. pylori characterise a genomic architecture within regions of high plasticity. This study provides a solid exploratory foundation for future functional research on the adaptive and pathogenic evolution of H. pylori.

Pathogens doi: 10.3390/pathogens15050460

Authors: Judit Henczkó Zsuzsa Kienle János Kádár Enikő Kádár-Hürkecz Katalin Tárnoki-Boross Panna Sütő Bernadett Pályi Ákos Tóth Katalin Kamotsay Zsuzsanna Molnár Zoltan Kis

Background: Brucellosis is a globally distributed zoonotic disease characterized by highly variable clinical manifestations that may mimic systemic autoimmune and inflammatory disorders. In Europe, where the incidence of brucellosis is relatively low, limited clinical awareness may contribute to delayed diagnosis and inappropriate management. In addition to zoonotic transmission, Brucella species are a well-recognized cause of laboratory-acquired infections (LAIs) among microbiology laboratory personnel. Methods: We report a case of laboratory-acquired Brucella abortus infection in a young woman presenting with undulant fever, arthralgia, systemic inflammation, elevated ferritin levels, and antinuclear antibody (ANA) positivity. Microbiological confirmation was achieved through serological testing (ELISA), repeat blood cultures, species-specific quantitative PCR, and whole-genome sequencing (WGS) followed by core genome multilocus sequence typing (cgMLST). Results: Initial laboratory evaluation revealed elevated C-reactive protein, mildly increased ferritin levels (146 ng/mL), abnormal liver enzyme levels, and rising ANA titers (from 1:160 to 1:320), raising suspicion of a systemic autoimmune disorder and prompting consideration of corticosteroid therapy. Although the initial blood culture was negative, subsequent molecular diagnostics and repeat cultures confirmed B. abortus infection. Epidemiological investigation suggested a possible occupational exposure in a diagnostic microbiology laboratory, consistent with a laboratory-acquired infection. Genomic analysis classified the isolate as sequence type 1 (ST1) and demonstrated zero allelic differences compared with the ST1 reference strain. Targeted antimicrobial therapy resulted in complete clinical recovery, supporting an infection-triggered immune response rather than primary autoimmunity. Conclusions: Acute brucellosis should be considered in the differential diagnosis of febrile syndromes accompanied by autoimmune-like laboratory abnormalities, even in low-incidence regions. This case highlights the diagnostic challenges posed by laboratory-acquired brucellosis and underscores the importance of early microbiological investigation and strict biosafety awareness in laboratory settings.

Pathogens doi: 10.3390/pathogens15050458

Authors: Māris Seņkovs Vizma Nikolajeva Luīze Rubene Kristians Jauga Līga Zemeca Inta Jakobija

The increasing demand for sustainable plant protection products has intensified interest in microbial biocontrol agents (BCAs). This study aimed to evaluate the antifungal activity of selected Streptomyces, Bacillus, and Trichoderma asperellum strains against phytopathogenic fungi and to assess their potential as BCAs under in vitro conditions. The antifungal activity of ten Streptomyces strains was first evaluated against Botrytis cinerea, Colletotrichum salicis, Fusarium oxysporum, and F. graminearum using a dual-culture assay. All isolates exhibited antifungal activity, with Streptomyces venezuelae MSCL 350 showing the strongest inhibition. In addition, the antifungal activity of T. asperellum MSCL 309 and three Bacillus strains was assessed against twelve Fusarium spp. isolates obtained from oats. T. asperellum demonstrated broad-spectrum inhibition, with growth inhibition ranging from 44.6% to 78.4%, primarily due to soluble metabolites, while volatile compounds showed no significant effect. Among the other tested Bacillus strains, only Bacillus subtilis MSCL 1441 exhibited antifungal activity, inhibiting all tested isolates. These results demonstrate strong strain-dependent antifungal activity and highlight T. asperellum MSCL 309, S. venezuelae MSCL 350, and B. subtilis MSCL 1441 as promising candidates for the development of environmentally friendly biocontrol agents.

Pathogens doi: 10.3390/pathogens15050459

Authors: Albert Gandurski Marta Tokaj Michał Jerzak Aleksandra Popławska-Ferenc Piotr Małkowski Monika Dybicz

Cystic echinococcosis (CE) is caused by a tapeworm of the Echinococcus granulosus s.l. species complex belonging to the Taeniidae family. CE affects more than 100 countries, including Poland, while remaining a significant public health threat to both humans and livestock. The aim of this study was to identify the genotypes responsible for cases of cystic echinococcosis in Poland by conducting molecular analysis of larvae isolated from Polish patients, and to investigate the diagnostic pathways leading to CE diagnosis. Between April 2023 and January 2025, tissue samples were collected from 10 patients following hepatectomy. Analysis of diagnostic pathways revealed that radiological findings followed by PCR or histopathological testing were sufficient to establish a reliable diagnosis of CE in 90% and 100% of cases, respectively. Serological tests showed lower sensitivity, reaching 86% for ELISA and 71% for Western blot. DNA extracted from all samples was used as the template in PCR to amplify and sequence the region of the mitochondrial NADH dehydrogenase 1 gene (nad1). PCR analysis confirmed presence of Echinococcus granulosus s.l. species in eight cases. All obtained nad1 sequences showed identity with the Echinococcus canadensis G7 (pig) strain. These results indicate that it remains the most frequent causative agent of human cystic echinococcosis in Poland.

Pathogens doi: 10.3390/pathogens15050457

Authors: Matías A. Dorsch Nicholas Crossland Fabio Del Piero Javier G. Nevarez Thomas N. Tully Maria S. Mitchell Mariano Carossino

Salmonella spp. is a major zoonotic pathogen. Although reptiles are mostly considered subclinical carriers, clinical disease may develop following immunosuppression. Clinical salmonellosis in reptiles has been extensively reported; however, the condition has been rarely described in bearded dragons (Pogona vitticeps). We retrospectively analyzed six cases of salmonellosis in bearded dragons and characterized the pathological, immunohistochemical, and bacteriological findings. Clinical signs and gross findings were mostly non-specific. Histological findings mainly consisted of fibrinonecrotizing enterocolitis (83.3%); necrotizing or granulomatous hepatitis (66.7%); pneumonia including bronchopneumonia or interstitial pneumonia in one case each (33.3%); tubulointerstitial nephritis with tubular necrosis (16.7%); and coelomitis (16.7%). Salmonella enterica subsp. houtenae was cultured in three cases (33.3%), whereas S. enterica subsp. enterica serovar Rissen, S. enterica subsp. enterica serovar Cotham, and S. enterica subsp. diarizonae were cultured in one case each. Intralesional bacteria were detected via immunohistochemistry in kidneys and colon in two cases (33.3%). The predominance of lesions in the intestines and liver likely reflects initial intestinal colonization followed by hematogenous dissemination to the liver. Hepatic lesions are thought to represent different stages along a continuum, progressing from acute necrosis to discrete granuloma formation. Renal and respiratory involvement was infrequent, as reported in other reptile species. Some of the isolated Salmonella subspecies (S. diarizonae and S. houtenae) are well-recognized causes of clinical disease in other reptile species but not previously identified in bearded dragons. This study provides a comprehensive pathological, immunohistochemical, and bacteriological characterization of salmonellosis in bearded dragons, thus raising awareness and assisting in the identification of this condition.

Pathogens doi: 10.3390/pathogens15050456

Authors: Duoxiao Zhang Jie Wang Zijin Zhao Yanqing Tie Jianing Wu Shihao Jiao Xingyu Liu Yuxin Wang Shijue Gao Mengchuan Zhao Pei Zhao Zhiqiang Han Xiaona Lyu Xinxin Shen Xuejun Ma Zhishan Feng

In this study, we developed a multiplex one-tube nested real-time fluorescent quantitative PCR (mONRT-PCR) assay integrated with a portable, fully automated nucleic acid point-of-care testing (POCT) platform for the detection of Haemophilus influenzae (H. influenzae), Listeria monocytogenes (L. monocytogenes), and Cryptococcus neoformans (C. neoformans) in cerebrospinal fluid (CSF). The assay enables nested amplification within a closed system using conventional primers and probes, thereby reducing operational complexity and minimizing contamination risk. Analytical evaluation demonstrated limits of detection of 100 copies/μL for H. influenzae and L. monocytogenes, and 101 copies/μL for C. neoformans using recombinant plasmids, as well as 10−7 to 10−6 ng/μL using genomic DNA. No cross-reactivity was observed when tested against a panel of 17 common non-target microorganisms encountered in clinical microbiology laboratories. In simulated CSF samples, the assay maintained detectable amplification at low pathogen concentrations. When implemented on the POCT platform, detection limits reached 5, 10, and 50 CFU/mL for the three pathogens, respectively. Clinical evaluation using 43 CSF samples showed almost perfect agreement with conventional qPCR (κ = 0.861, p < 0.001). Notably, additional C. neoformans detections were observed by mONRT-PCR-POCT compared with qPCR, suggesting improved sensitivity under clinical conditions. The assay yielded results within approximately 1 h and 47 min. These findings indicate that the proposed assay provides a rapid, sensitive, and integrated approach for meningitis pathogen detection, while maintaining a practical balance between analytical performance and operational simplicity. Further validation in larger cohorts is warranted.

Pathogens doi: 10.3390/pathogens15050455

Authors: Maryam Gul Sajid Ali Abdul Rehman Muhammad Qasim Roomana Ali Jody E. Phelan Aiman Waheed Sajjad Ahmad Mubbashir Hussain Susana Campino Taane G. Clark Taj Ali Khan

Background: Resistance to first-line anti-tuberculosis drugs in Mycobacterium tuberculosis represents a significant public health challenge, particularly in high-burden tuberculosis (TB) settings such as Pakistan, where multidrug-resistant (MDR) forms further complicate disease control efforts. Drug resistance is primarily associated with mutations in rpoB, inhA, katG, embA, embB, embC, and pncA. The emergence of novel, region-specific variants underscores the urgent need for integrating genomic surveillance into routine TB diagnostics and regional control programs. This study aimed to identify the spectrum of mutations contributing to first-line drug resistance in MDR-TB isolates from Khyber Pakhtunkhwa, Pakistan. Methods: Whole-genome sequencing was performed on 16 clinical isolates (12 MDR and 4 drug-susceptible) to identify resistance-associated mutations in rpoB, inhA, katG, embA, embB, embC, and pncA. Detected variants were interpreted using the World Health Organization (WHO) mutation catalogue to determine their association with drug resistance. Phylogenetic relationships were inferred using the Bacterial and Viral Bioinformatics Resource Center (BV-BRC) platform. Results: A total of 16 M. tuberculosis isolates were analyzed to evaluate resistance to first-line anti-tuberculosis drugs. In rpoB, 76 distinct variants were identified, including canonical mutations such as Ser450Leu and His445Arg, as well as a potentially novel substitution, Ser431Phe, predicted to confer high-level rifampicin resistance. The katG and inhA genes harbored 24 and 27 mutations, respectively, including well-characterized substitutions such as Ser315Thr and Ala114Glu, which are strongly associated with isoniazid resistance. Mutations in embA and embB were linked to ethambutol resistance, with several variants localized within conserved transmembrane domains critical for drug interaction. Phylogenetic analysis revealed substantial genetic diversity and evidence of local transmission among MDR-TB isolates. Conclusions: This study suggests that the genetic landscape of drug resistance in M. tuberculosis is highly dynamic in endemic regions. The findings highlight the importance of integrating region-specific mutation profiles into molecular diagnostic frameworks to enhance early detection, guide individualized therapeutic interventions, and strengthen strategies aimed at controlling the transmission of MDR-TB.

Pathogens doi: 10.3390/pathogens15050454

Authors: Marília Mazzi Moraes Natália de Godoy Eduardo Maffud Cilli Paulo Ricardo da Silva Sanches

Chikungunya virus (CHIKV) infection presents a wide spectrum of clinical outcomes, ranging from mild self-limiting disease to severe and fatal manifestations, which are influenced by both host and viral factors. Animal models are essential for elucidating CHIKV pathogenesis and for preclinical evaluation of antiviral strategies; however, a well-characterized model evaluating the effect of different viral doses in AG129 mice remains limited. In this study, we investigated the clinical, virological, and pathological outcomes of CHIKV infection in male AG129 mice inoculated intraperitoneally with different viral doses (10, 100, and 1000 PFU/mL) of a Brazilian strain belonging to the East/Central/South African (ECSA) lineage. Lower-dose inoculation (10 PFU/mL) resulted in a milder disease course, characterized by transient viremia, limited tissue viral dissemination, minimal histopathological alterations, partial survival, and viral clearance. In contrast, higher doses (≥100 PFU/mL) led to rapid systemic viral dissemination, severe histopathological damage in the spleen, liver, and kidneys, and uniform lethality. Viral RNA was detected in serum and multiple organs in a time-dependent manner, with limited differences among inoculum doses in most tissues. Notably, dose-related differences were observed in specific compartments and time points, particularly in hind-limb muscles at early time points and in serum at later stages.

Pathogens doi: 10.3390/pathogens15050453

Authors: Claudio Marcello Melini Mariana Kikuti Xiaomei Yue Cesar A. Corzo

Porcine reproductive and respiratory syndrome virus (PRRSV)-positive pigs can be exposed to high concentration of gases, such as ammonia and hydrogen sulfide when manure and urine stored in the pits beneath them is agitated and pumped. Such acute exposure can lead to adverse health effects such as respiratory system irritation. This study aimed to explore whether PRRS-positive growing pigs experience changes in viremia detection after manure pit agitation and pumping has been performed. To address this objective, two PRRS-positive growing pig farms were conveniently selected and visited twice during the week before and after manure agitation and pumping. Blood samples were collected to assess detection of viremia, using reverse transcription-polymerase chain reaction (RT-PCR). A logistic regression model was used to evaluate serum detection of PRRSV before and after the manure management event, accounting for pig age. Although PRRSV was detected in the serum of some pigs, under the conditions of the study, there were no statistically significant changes that would indicate that viremia detections change after the pigs had been exposed to barn manure pit agitation and pumping.

Pathogens doi: 10.3390/pathogens15050452

Authors: Laura Veschetti Angela Sandri Giulia Maria Saitta Marzia Boaretti Paola Melotti Cristina Cigana Alessandra Bragonzi Maria M. Lleò Giovanni Malerba Caterina Signoretto

Achromobacter spp. are opportunistic pathogens in people with cystic fibrosis (PwCF), yet the role of the upper airways in their persistence and adaptation remains poorly understood. We investigated whether the sinonasal compartment may act as reservoir and evolutionary niche for Achromobacter spp. during airway infection. Twenty-two isolates obtained from paired nasal lavage and sputum samples of seven PwCF were analysed by whole-genome sequencing. Within each PwCF, identical clone types were detected in both airway compartments, supporting bacterial exchange between upper and lower airways. Despite clonal relatedness, substantial genomic diversification was observed between paired isolates. Genomic signatures indicative of elevated mutation rates were detected in a high number of isolates (73%) and in both airway compartments, highlighting widespread genomic diversification across the respiratory tract. Mobilome analysis revealed compartment-specific variations in insertion sequences, prophages, and integrative elements, suggesting genome plasticity. Additionally, mutation in an aspartate kinase gene was consistently associated with loss of biofilm formation in vitro, highlighting a potential link between this pathway and biofilm phenotype. Overall, our findings indicate that upper and lower airways represent interconnected but partially independent ecological niches where Achromobacter populations can diverge during colonization, supporting the view that both compartments contribute to their persistence and evolution in CF airways.

Pathogens doi: 10.3390/pathogens15040451

Authors: Isabel Lagos Edith Pérez de Arce Ilaria Faggiani Ferdinando D’Amico Alessandra Zilli Federica Furfaro Sara Massironi Clelia Cicerone Virginia Solitano Tommaso Lorenzo Parigi Laurent Peyrin-Biroulet Silvio Danese Mariangela Allocca

Inflammatory bowel diseases (IBDs), including ulcerative colitis (UC) and Crohn’s disease (CD), are consistently associated with alterations in gut microbial communities, although the extent and characteristics of these alterations vary across studies, supporting a potential role of the microbiota in disease pathogenesis and therapeutic modulation. We conducted a systematic review to synthesize current evidence on microbiota alterations in IBD and the clinical application of fecal microbiota transplantation (FMT). A total of 118 studies were included (76 focused on microbiota profiling and 42 evaluated FMT as therapy). Across heterogeneous study designs and microbial characterization methods, reduced microbial diversity was the most consistently reported alteration, generally more pronounced in CD than in UC. Depletion of Faecalibacterium prausnitzii—a key butyrate producer with anti-inflammatory properties—was commonly reported, often accompanied by functional impairment in short-chain fatty acid production. Microbial patterns were frequently associated with mucosal inflammation and varied across disease phenotypes; these patterns have been increasingly explored as predictors of treatment response and relapse, although mechanistic interpretation remains limited and causal relationships are difficult to establish. Evidence from randomized controlled trials suggests potential efficacy of FMT in UC, particularly with intensive or repeated protocols, whereas data in CD remain limited and heterogeneous, with signals of benefit often appearing transient. FMT was generally well tolerated, but long-term safety data remain scarce. Emerging multi-omic approaches are reshaping the field by integrating taxonomic and functional insights, with potential implications for risk stratification, diagnosis, prognosis, and therapeutic optimization. Further standardized, longitudinal, and mechanistically oriented studies are required to translate microbiome research into clinically actionable strategies in IBD.

Pathogens doi: 10.3390/pathogens15040450

Authors: Shinsuke Nakajima Masaki Hino Norihiro Takenouchi Yoshihisa Yamano Makoto Yamagishi Tokifumi Odaka Fhahira Rizkhika Admadiani Cecile Faye Kaoru Uchimaru Jun-Ichi Fujisawa Kazu Okuma

Human T-cell leukemia virus type 1 (HTLV-1)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is a chronic inflammatory disease driven by HTLV-1-infected CD4+ T cells; however, the phenotypic and functional characteristics of disease-associated T-cell subsets remain incompletely understood. We analyzed samples using flow cytometry (n = 3–5 per group) and RNA-seq (n = 13), focusing on CADM1highCD4+ T cells enriched for HTLV-1-infected cells to evaluate a transferrin receptor (TfR)-expressing subset. TfR+CADM1highCD4+ T cells were detected in both asymptomatic carriers and patients with HAM, but their frequency among CD4+ T cells was higher in HAM patients. These cells exhibited a Treg-like phenotype with higher Foxp3 and CTLA-4 expression than TfR− cells and showed increased Ki-67 positivity, consistent with proliferation. Despite this phenotype, they produced interferon-γ, indicating inflammatory potential, while Foxp3 expression was lower in HAM patients than in asymptomatic carriers, suggesting a more inflammatory phenotype. Furthermore, TfR transcript levels (RNA-seq TPM) correlated with clinical indicators of disease activity, including neopterin and CXCL10 protein levels, and the Osame motor disability score. Collectively, these findings suggest that TfR identifies a proliferative, Foxp3-low, Treg-like inflammatory CD4+ T-cell subset that is associated with disease activity in HAM.

Pathogens doi: 10.3390/pathogens15040449

Authors: Francesco Nappi

Carbapenem-resistant Acinetobacter baumannii infections pose a significant challenge due to their severity and the poor prognoses they often result in, particularly in cases where there are risk factors present. The United States (US) Centers for Disease Control and Prevention (CDC) identified carbapenem-resistant Acinetobacter baumannii (CRAB) infections as a threat to human health. The World Health Organization (WHO) has classified it as a top priority for research. In 2023, the US FDA approved sulbactam–durlobactam for treating certain A. baumannii infections. As of 2024, this combination is designated as the preferred treatment strategy by the Infectious Diseases Society of America (IDSA) for infections due to carbapenem-resistant A. baumannii. In this therapeutic review, the preclinical and clinical data relevant to this regulatory decision were analyzed. This in-depth analysis will provide a comprehensive overview of the complex subject matter. It should be observed that carbapenem-based combination therapy is indicated for carbapenem-resistant A. baumannii.

Pathogens doi: 10.3390/pathogens15040448

Authors: Tianyu Lei Mengtao Yu Panjing Lv Hui Deng Di Yang Kaijie Li Yan Li

The protozoan parasite Leishmania mexicana serves as a widely used model for studying trypanosomatid biology, yet the demand for stable, high-intensity fluorescent tools for precise subcellular protein localization remains unmet. In this study, we developed a versatile molecular toolbox by re-engineering the pLEXSY-hyg2.1 vector to express mNeonGreen (mNG), a next-generation fluorophore with superior brightness and photostability. Using a modular cloning strategy, we introduced a customized multiple cloning site (MCS) upstream of the mNG sequence to facilitate seamless N-terminal tagging of target proteins. The construct was integrated into the 18S rRNA locus via homologous recombination to ensure stable, constitutive expression. As a proof-of-concept, we fused a flagellar marker to the mNG reporter, resulting in a transgenic line exhibiting robust and specific subcellular fluorescence without compromising cellular fitness. Our results demonstrate that this integration-based system provides a highly efficient and stable platform for visualizing protein distribution within Leishmania. This tool significantly simplifies the generation of reporter strains and will facilitate high-resolution imaging studies of parasite organelle dynamics and functional genomics.

Pathogens doi: 10.3390/pathogens15040447

Authors: Daniel Pérez-Prieto Svetlana Karbysheva Andrej Trampuz Oscar Fariñas Joan C. Monllau Ferran Corcoll

In the original publication [...]

Pathogens doi: 10.3390/pathogens15040446

Authors: Alessandra Picelli Emma Papini Guglielmo Bonaccorsi Angela Bechini Fabiola Berti Sara Boccalini Paolo Bonanni Manuela Chiavarini Claudia Cosma Chiara Lorini Cristina Salvati Valentina Saviozzi Patrizio Zanobini Marco Del Riccio Saverio Caini

Background: Human rhinoviruses (HRVs) exhibit a global circulation characterized by prolonged epidemics and a less concentrated seasonal distribution compared with other respiratory viruses. In this study, we describe the timing, amplitude and duration of HRV epidemics on a global scale, analyzing seasonal patterns in relation to geographic latitude. Methods: HRV surveillance data reported to WHO FluNet from 2016 to 2025 were analyzed. Epidemic peak timing, amplitude and duration were estimated as a function of geographic latitude using harmonic analyses, with a comparison between the pre-pandemic (2016–2019) and post-pandemic (2021–2025) periods. Results: During the study period, 432,399 HRV detections were reported to WHO FluNet across 50 countries. Among these, 24 countries met the predefined criteria for seasonal analysis. Epidemic peak timing showed differences consistent with latitude, with peaks occurring in late autumn and winter in the Northern Hemisphere, during the central months of the year in the Southern Hemisphere, and greater temporal variability in the intertropical belt. Peak amplitude showed marked heterogeneity across countries (median 68.2%, range 28.1–96.7%), while epidemic duration indicated prolonged circulation (median 31 weeks, range 5–48 weeks). A secondary seasonal peak was identifiable in most countries, further supporting the relatively diffuse seasonal profile of HRV circulation. Comparison between the pre- and post-pandemic periods showed largely stable peak timing in most countries, alongside heterogeneous changes in peak amplitude. Conclusions: HRV is characterized by prolonged and weakly concentrated seasonal activity, with epidemic circulation often extending over several months. Despite major epidemiological perturbations during the COVID-19 pandemic, the timing of seasonal peaks remained largely stable across countries, highlighting the epidemiological resilience of HRV and the need for continuous, pathogen-specific surveillance.

Pathogens doi: 10.3390/pathogens15040445

Authors: Miłosz Tkaczyk Katarzyna Sikora

Horse chestnut (Aesculus hippocastanum L.) is a widely planted ornamental and urban tree valued for its aesthetic and ecological functions. In recent years, declining health of horse chestnut in urban environments has been increasingly reported, often associated with a complex of biotic and abiotic stressors. During a health survey of A. hippocastanum trees growing along an urban road corridor in Warsaw, Poland, extensive bark necrosis and branch dieback were observed. The aim of this study was to identify the causal agent of these symptoms using morphological, cultural, molecular (ITS rDNA), and pathogenicity tests under controlled conditions. Fungal isolates were obtained from necrotic tissues and were consistently identified as Kalmusia variispora based on ITS sequence analysis (99.0–99.6% similarity to GenBank references) and characteristic morphology. Pathogenicity tests fulfilled Koch’s postulates, reproducing necrotic lesions and cambial damage similar to those observed in the field. To our knowledge, this is the first documented report worldwide of K. variispora infecting A. hippocastanum. The findings expand the known host range of this opportunistic Didymosphaeriaceae species and highlight its potential role in bark and wood disease complexes of urban trees. Further research is needed to assess its distribution, genetic diversity, and epidemiological significance in urban forest ecosystems.

Pathogens doi: 10.3390/pathogens15040444

Authors: Anna O. Busbee Aryashree Arunima James E. Samuel Erin J. van Schaik

C. burnetii (Cb) is an obligate intracellular bacterial pathogen that replicates within alveolar macrophages following aerosol infection. Unlike most intracellular bacteria, Cb establishes a lysosome-derived replicative niche (Coxiella-containing vacuole or CCV) through the action of its Type IVB secretion system (T4BSS). This system translocates a large repertoire of effector proteins into the host cytoplasm after phagosome acidification. These effectors interfere with diverse signaling pathways to co-opt host processes, such as vesicle trafficking, ubiquitylation, gene expression and lipid metabolism, promoting pathogen survival without triggering robust proinflammatory signaling or host cell death pathways. This effector-triggered immune silencing is particularly unique given the central role of macrophages as innate immune sentinels. In this review, we examine Cb T4BSS effectors that have been characterized as central determinants of innate immunity modulation. We discuss innate immune sensing pathways potentially engaged during infection, including Toll-like receptors, NOD-like receptors, RIG-I-like receptors, inflammasomes, and interferon signaling pathways, and highlight evidence indicating that these pathways are actively suppressed. Emphasis is placed on effector-mediated regulation of NF-κB signaling, type I interferon responses, and inflammasome activation. Finally, we address unresolved questions related to effector-triggered immunity, redundancy in immune suppression, and discrepancies between in vitro and in vivo infection models.

Pathogens doi: 10.3390/pathogens15040443

Authors: Gaetano Scotto Vincenzina Fazio Ali Muhammed Moula Sri Charan Bindu Bavisetty Alessia Franza Salvatore Massa

Nipah virus (NiV) is a highly pathogenic zoonotic paramyxovirus responsible for sporadic outbreaks of severe disease with high case fatality rates in South and Southeast Asia. Human infection occurs through spillover from natural reservoirs, primarily fruit bats, or via human-to-human transmission, and is characterized by a broad clinical spectrum ranging from asymptomatic infection to acute respiratory disease and fatal encephalitis. Following entry via ephrin-B2 and ephrin-B3 receptors, NiV exhibits marked endothelial and neuronal tropism, leading to systemic vasculitis, disruption of the blood–brain barrier, and direct infection of the central nervous system. Disease progression is driven by a complex interplay between viral replication strategies and host immune responses. NiV effectively counteracts innate immunity through multiple viral proteins that inhibit interferon signaling, while simultaneously inducing dysregulated inflammatory responses that contribute to tissue damage and multi-organ failure. Neurological involvement represents the most severe manifestation, often resulting in acute or relapsing encephalitis with long-term sequelae among survivors. Despite the severity of the disease, no licensed antiviral therapies or human vaccines are currently available. Therapeutic development has focused on neutralizing monoclonal antibodies targeting viral glycoproteins and small-molecule antivirals that inhibit viral RNA synthesis, both of which show promising results in preclinical models, but remain limited by timing and translational challenges. In parallel, several vaccine platforms—including viral vectors, mRNA-based constructs, and recombinant protein subunits—have advanced to early-phase clinical trials, demonstrating encouraging immunogenicity. Beyond biomedical interventions, effective outbreak containment relies on integrated public health strategies. The “Kerala model” highlights the importance of rapid case identification, isolation, contact tracing, and community engagement within a One Health framework to mitigate transmission and reduce mortality. This review synthesizes the current knowledge on NiV pathogenesis, immune evasion, clinical manifestations, and emerging therapeutic and vaccine strategies, while highlighting critical gaps and future directions for improving the preparedness and response to this high-consequence emerging pathogen.

Pathogens doi: 10.3390/pathogens15040442

Authors: Albert Farrugia Laurence Corash Raymond Goodrich Leni von Bonsdorff

Therapeutics derived from donated blood or its constituents are classifiable into blood components and plasma derivatives. The latter are defined as medicines/drugs/pharmaceuticals produced from the industrial fractionation of thousands of pooled plasma donations and characterised with relative precision to a pre-defined specification through sampling of a homogenous pharmaceutical batch. The former are defined as components/biologicals produced using relatively simple (but increasingly complex) technologies in blood centres from single or small pools of isolated components from whole blood and are pre-specified through regulatory standards with relatively wide limits because of the inherent biologic variability of individual donors. This review discusses the evolution of technology to reduce the risk of pathogen transmission by blood-derived therapeutics, assess the state of the approved technologies for pathogen-reduced blood components, and examine the features of the blood-provider and regulatory framework globally that have shaped, and in some instances impeded, the implementation of component pathogen reduction to an extent equivalent to that achieved for plasma derivatives. The ensuing risks to the public’s confidence in the blood supply are discussed, and remedial actions are proposed. The features of a new paradigm for blood safety are outlined.

Pathogens doi: 10.3390/pathogens15040438

Authors: Hernán D. Nievas Camila Aurnague Elisa Helman Raúl E. Iza Magdalena Costa Oliver Mounsey Matthew B. Avison Lucía Galli Fabiana A. Moredo

The occurrence of Escherichia coli resistant to high-priority critically important antimicrobials (HPCIA) in the food chain is a growing concern for food safety and public health. This study aimed to evaluate whether HPCIA-resistant E. coli isolated from pork and chicken meat at retail markets in La Plata, Buenos Aires, Argentina, exhibit source-associated genomic differentiation through whole-genome sequencing. The isolates displayed a polyclonal population structure, encompassing multiple phylogenetic groups and sequence types. Virulence gene profiles were highly diverse, with chicken-derived isolates harbouring a substantially higher number of virulence genes than pork isolates. Notably, one pork isolate carried a complete set of virulence genes characteristic of diarrheagenic E. coli. Single Nucleotide Polymorphism-based phylogenetic analysis revealed several closely related subclusters, including strains recovered from both pork and chicken meat from the same retail markets, suggesting recent clonal sharing or cross-contamination at the point of sale. These findings highlight the circulation of genetically diverse HPCIA-resistant E. coli in retail meat, underscoring the potential public health risk and the importance of monitoring resistance and virulence determinants throughout the food production chain.

Pathogens doi: 10.3390/pathogens15040441

Authors: Benjamin H. Nguyen Elise Stanley Victoria K. Baxter Diane E. Griffin

Alphaviruses are mosquito-borne viruses that can infect the central nervous system (CNS) and cause encephalomyelitis, which is a rare but dangerous complication from infection. In mice, this can be studied in a model of infection with Sindbis virus (SINV), which infects neurons and causes neurological disease. Due to the non-renewable nature of neurons, the immune response in the CNS is specialized to prevent neuronal damage or death, even if they are infected. Therefore, insights into the nuances of antiviral immunity in the CNS provide a better understanding of disease pathogenesis and mechanisms of recovery. Type I interferons (IFNs) are critically important for survival; they are an innate antiviral defense mechanism that consists mainly of IFNα and IFNβ. Although both use the same receptor, type-specific differences between IFNα and IFNβ have been described in other contexts. To this end, Ifnb−/− mice were used to elucidate the role of IFNβ in recovery from alphavirus encephalomyelitis. IFNβ-deficient mice have intact IFNα expression and downstream signaling, but symptomatic disease occurs earlier and is more severe. This is accompanied by increased virus replication in the early stages of infection. Microgliosis is reduced in Ifnb−/− mice compared to wildtype, but inflammatory cytokine/chemokine levels are higher and associated with alterations in monocyte and NK cell recruitment into the CNS. Ifnb−/− mice have no deficiencies in the expression of factors known to be required for viral clearance. Therefore, IFNβ modulates the early stages of the immune response and facilitates restriction of virus replication, contributing to delayed disease onset.

Pathogens doi: 10.3390/pathogens15040440

Authors: Elianne Piloto-Sardiñas Islay Rodríguez Huarrisson Azevedo Santos Patrícia Gonzaga Paulino Belkis Corona-González Alejandro Cabezas-Cruz

Ticks harbor complex microbial communities composed of symbionts, commensals, and tick-borne pathogens (TBPs). Together, these microorganisms form the tick holobiont. Within this system, the tick’s physiological architecture structures microbial communities by distributing microorganisms across distinct tissues. This compartmentalization creates spatially distinct ecological niches, which in turn shape how microbial communities assemble and interact. In this review, we integrate ecological theory with current knowledge of tick microbiome research to examine how pathogen–pathogen and pathogen–microbiome interactions emerge within these tissue-structured microbial communities. We first outline how baseline ecological filters, including tick species, developmental stage, tissue identity, vertical transmission, and environmental context, shape the microbiome configuration through community assembly processes. We then examined how TBPs, as high-impact colonizers, can further modify microbial networks by altering host-mediated selective pressures, influencing interaction topology, and reshaping community stability. Based on these observations, we propose a dual selective pressure framework in which (i) baseline ecological structuring processes and (ii) pathogen-associated selective pressures interact to determine the microbial network configuration and functional outcomes within the tick holobiont. These interacting forces may drive shifts in diversity, modularity, keystone taxa emergence, and network resilience, ultimately influencing vector competence. This review frames the microbial communities within the tick holobiont as spatially structured ecological systems shaped by multilevel selective pressures. This conceptual foundation provides a coherent framework for understanding microbial interactions in arthropod vectors and highlights avenues for mechanistic research and microbiome-based strategies to mitigate tick-borne diseases.

Pathogens doi: 10.3390/pathogens15040439

Authors: Berenice Munguía-Ramírez Betsy Armenta-Leyva Luis Giménez-Lirola Yanqi Zhang Bailey Arruda Giovana Ciacci-Zanella Jeffrey Zimmerman

Influenza A virus (IAV) surveillance in swine relies heavily on molecular detection, yet RNA stability in diagnostic specimens such as oral fluids can be rapidly compromised when cold-chain conditions are not maintained. This pilot study evaluated the ability of four molecular-grade carbohydrates (20% trehalose, sorbitol, sucrose, and mannitol) and two commercial nucleic acid stabilizers (PrimeStore® MTM and RNAlater®) to preserve RT-qPCR-detectable IAV RNA in swine oral fluids exposed to field-relevant stress conditions. Oral fluid samples collected from pigs experimentally infected with H1N2 (Study 1: n = 150; DPIs 2, 3, 4) or with H1N2 and H3N2 (Study 2: n = 58; DPI 5) were subjected to storage at 25 °C for up to 144 h (Study 1) or 2, 5, 10, or 15 freeze–thaw cycles (Study 2), with DPIs (Study 1) or subtypes (Study 2) serving as biological replicates, given the limited sample size. IAV detection was quantified as efficiency standardized Cq values (ECq) and analyzed using a linear mixed-effects model. Overall, both carbohydrates (trehalose, sorbitol, sucrose) and commercial stabilizers maintained higher ECq values than untreated oral fluids under both thermal and freeze–thaw stress conditions. Due to the limited sample size, these findings should be interpreted cautiously, yet they demonstrate the potential utility of carbohydrates as a low-cost, non-inactivating alternative for stabilizing IAV RNA in field-collected oral fluids.

Pathogens doi: 10.3390/pathogens15040437

Authors: Ade Xin Ning Tan Ilyas Hussin Chia Yin Chong Matthias Maiwald Terri Xiao-Bei Chiong Natalie Woon Hui Tan

Lyme disease is the most common reported vector-borne disease in North America and is also highly prevalent across Europe. Although tick-borne diseases are uncommon in Singapore, there remains a risk of imported tick-borne diseases among travelers from endemic regions. We present a case series of three pediatric patients with imported Lyme disease managed at a tertiary children’s hospital in Singapore, illustrating the varied clinical presentations of Lyme disease in children. One child developed meningitis following prior antibiotic therapy for Lyme disease, although causality cannot be definitively established. This series aims to highlight key diagnostic considerations and management principles relevant to clinicians practicing in non-endemic regions.

Pathogens doi: 10.3390/pathogens15040436

Authors: Stephen A. Klotz Richard E. Sobonya Peter N. Lipke

Candida albicans is a ubiquitous commensal fungus that may be lethal once it gains access to the bloodstream, following a breach in protective barriers such as skin or gut lining. Intravenous injection of C. albicans (4.5 × 104 yeasts/gm of mouse) leads reproducibly to systemic infection with a median survival of about 75 h. We studied the effects of two human innate immune effectors on the course of systemic infections. The soluble human pentraxin serum amyloid P component (hSAP) retards death in murine disseminated candidiasis. In contrast, another soluble pentraxin, human C-reactive protein (hCRP), hastens death. To examine the pathological basis for these differences, necropsies were performed, and the right kidney was removed for study. Candidiasis caused abundant collagen deposition (the precursor to fibrosis) and loss of contrast between the kidney medulla and cortex. Daily administration of subcutaneous hSAP following the intravenous injection of C. albicans preserved the discrete histological difference between cortex and medulla and lessened host collagen deposition. Yeasts and hyphae within abscesses were decorated with hSAP. Contrastingly, kidneys from animals administered C. albicans and hCRP showed extensive collagen deposition and loss of the boundary between the cortex and the medulla of the kidney. hCRP did not bind to fungi but bound to damaged tissue surrounding abscesses, leading to a more destructive infection with loss of tissue. Staining cells with antibodies to CD45 (to detect T-lymphocytes, myelocytes, monocytes, and macrophages) and antibodies to Ly-6G (neutrophils, and granulocytes) showed that hSAP retarded infiltration of inflammatory cells into diseased areas. The results are consistent with the hypothesis that early administration of hSAP represses the migration of inflammatory cells, dampens the production of collagen by fibroblasts, and dampens the overall immune response of the host to infection. In doing so, hSAP prolonged life, whereas hCRP facilitated the infectious process and hastened death.

Pathogens doi: 10.3390/pathogens15040435

Authors: Deborah Cruciani Manuela Papini Luigi Pisano Roberta Calcaterra Donatella Pietrella Tommaso Galeotti Paolo Fazii Antonia Meloscia Martina Torricelli Marco Di Domenico Alessandro Fiorucci Sara Spina Silvia Crotti

Trichophyton indotineae is an emerging dermatophyte associated with extensive, chronic, recalcitrant, and frequently terbinafine-resistant dermatophytosis worldwide. In this study, 30 T. indotineae strains isolated in Italy were investigated. The isolates were obtained from patients originating from Asian countries, from patients from other countries, and from Italian patients who reported no travel outside Italy in the preceding years. Clinical isolates were identified by internal transcribed spacer (ITS) sequencing and analyzed to assess the occurrence and molecular basis of terbinafine resistance. Terbinafine resistance was detected in 18 strains (60%) using a real-time PCR assay. Sequencing of the squalene epoxidase (SQLE) gene revealed mutations associated with resistance, including L393S in nine strains and F397L in another nine strains. NGS analysis confirmed two terbinafine-resistant strains carrying the L393S and F397L mutations, respectively, and detected the A448T mutation in one terbinafine-susceptible strain. These findings demonstrate the presence of terbinafine-resistant T. indotineae across five regions of Italy and confirm the occurrence of SQLE mutations previously linked to antifungal resistance. Data obtained also support a link with endemic Asian areas, other than suggesting the possible occurrence of autochthonous transmission in Italy.

Pathogens doi: 10.3390/pathogens15040434

Authors: Kumar Sivasubramanian Rudrappan Raj Bharath Leela Kakithakara Vajravelu Madan Kumar D Jayakrishna Pamarthi

Background: Dengue fever is one of the most common mosquito-borne viral infections, with severe cases characterized by plasma leakage, hemorrhage, and multi-organ involvement. Identification of dengue serotypes and reliable biomarkers is essential for predicting disease progression and guiding timely interventions. Methods: This prospective cohort study was conducted at a super-speciality tertiary care hospital in southern India from July 2024 to July 2025. A total of 69 patients presenting with dengue warning signs were included in the study. Patients were categorized into the severe dengue group (n = 25) and non severe dengue group (n = 44). Clinical data, laboratory findings, dengue serotype, and serial serum samples collected on Days 1, 4, and 8 were analyzed to evaluate the predictive and monitoring efficacy of Interleukin-6 (IL-6) and Interleukin-8 (IL-8), and followed up till discharge. Results: Out of 69 dengue patients with warning signs, 32 dengue-positive patients were serotyped, which included DEN V-1 (31.3%), DEN V-2 (31.3%), DEN V-3 (15.6%), DEN V-4 (18.8%), and mixed DEN V-(2 + 3) (3.1%). Severe dengue patients exhibited a higher frequency of secondary dengue infection (IgG) than primary dengue infection (88% vs. 12%), with statistically significantly higher packed cell volume, hemoglobin levels, high AST levels, and prolonged activated partial thromboplastin time, as well as lower platelet counts and albumin levels. Platelet transfusion was given to 35 dengue patients, which had also resulted in significant length of stay in hospital in comparison to non-transfused patients. IL-6 and IL-8 levels were significantly elevated in severe dengue patients when compared to non-severe dengue patients on Day 1 and Day 4, followed by a decline on Day 8, corresponding with clinical recovery. However, the elevated IL-8 levels were observed to be significantly associated with longer hospital stays, indicating its potential role as an early predictor of disease progression. Conclusions: The observed co-circulation of multiple serotypes reflects the hyper-endemic pattern reported across India. Early measurement of these cytokines IL-6 and IL-8 helps distinguish severe from non-severe dengue among patients presenting with warning signs. IL-6 and IL-8 may have potential as biomarkers for disease severity. However their role in guiding platelet transfusion requires further investigation in non-severe cases and prioritizing timely management for those at higher risk of severe disease.

Pathogens doi: 10.3390/pathogens15040433

Authors: Xiaofei Huang Anqiang Zhang Dalin Wen He Li Ling Zeng

Reduced levels of vitamin D are associated with increased incidence and mortality of sepsis. Nonetheless, the effectiveness of vitamin D supplementation in improving sepsis patients’ outcomes continues to be debated. In this research, which was conducted as a retrospective cohort analysis, data obtained from the Medical Information Mart for Intensive Care IV (MIMIC-IV 3.0) were used. The focus of the study was on vitamin D3 administration to sepsis patients while in the ICU. The primary outcome measurement was 28-day ICU mortality, with secondary outcomes of mechanical ventilation duration, percentage of patients receiving mechanical ventilation, and ICU stay length. The Kaplan–Meier curve analysis, Cox regression analysis, and subgroup analyses were performed to explore the link between vitamin D3 supplementation and sepsis prognosis. A 1:1 propensity score matching (PSM) approach was used to strengthen the reliability of the results. Before matching, the cohort comprised 28,524 patients, which was reduced to 4,856 after PSM. The analysis revealed that vitamin D3 supplementation was associated with a lower 28-day ICU mortality rate (HR = 0.71, 95% CI: 0.64–0.78, p < 0.001). Kaplan–Meier curve analysis revealed significantly greater survival probabilities in the group receiving vitamin D3 than in the group not receiving vitamin D3 (p < 0.001). Subgroup analysis showed that total cumulative exposure to vitamin D3 was more strongly associated with 28-day ICU mortality (p < 0.001), whereas daily dose and dosing frequency showed no significant association. The results after PSM and subgroup analysis were consistent with those of the original cohort study, further confirming the robustness of the results. Overall, vitamin D3 supplementation is associated with lower 28-day ICU mortality and better outcomes in patients with sepsis. However, given the retrospective observational design, large-scale prospective randomized controlled trials are warranted to validate these observational associations and establish causal effects.

Pathogens doi: 10.3390/pathogens15040432

Authors: Jae-Yeon Choi Pallavi Singh Choukri Ben Mamoun

Polyamines, putrescine, spermidine and spermine, are essential polycationic metabolites present in all eukaryotic cells, where they regulate fundamental processes including nucleic acid stabilization, translation, and stress responses. Spermidine synthase (SPDS), a member of the aminopropyltransferase (APT) family, catalyzes the transfer of an aminopropyl group from decarboxylated S-adenosylmethionine (dc-SAM) to putrescine to form spermidine. Although genomic analyses predict the presence of SPDS homologs in multiple fungal species, polyamine biosynthesis has not been experimentally characterized in the multidrug-resistant fungal pathogen Candidozyma auris. Here, we report the biochemical and functional characterization of the C. auris spermidine synthase, CauSpe3. The CauSPE3 gene complemented a Saccharomyces cerevisiae spe3Δ mutant demonstrating conserved function in vivo. Recombinant CauSpe3 was expressed in Escherichia coli, purified and analyzed using the fluorescence-based DAB-APT assay, which uses 1,2-diacetylbenzene (DAB) for polyamine detection. CauSpe3 catalyzed efficient conversion of putrescine to spermidine in the presence of dc-SAM, with Khalf values of 65.5 ± 7.11 µM for putrescine and 66.9 ± 2.09 µM for dc-SAM, and Vmax values of 7.1 ± 0.57 and 7.9 ± 0.12 nmol·µg−1·min−1, respectively. A catalytic-site mutant and heat-inactivated enzyme showed no detectable activity, and product formation was confirmed by means of thin-layer chromatography and mass spectrometry. These findings establish CauSpe3 as a functional spermidine synthase.

Pathogens doi: 10.3390/pathogens15040431

Authors: Wenxian Wen Qin Du Shuhan Li Youmin Yang Xianding Wang Shasha Li Yujia Li Shilin Li Chunhui Yang He Xie Xiaoqiong Duan Limin Chen

We previously demonstrated that HBV promotes liver fibrosis through the enhanced production of pyruvate. Pyruvate kinase M2 (PKM2), a key enzyme in pyruvate metabolism, plays an important role in liver fibrogenesis. Recently, lactylation of PKM2 has been identified, which contributes to stabilize its catalytically active tetrameric conformation. Therefore, we hypothesize that PKM2 lactylation is involved in the regulation of HBV-induced liver fibrosis. In this study, we found that sera lactate levels were increased in CHB patients and HBV-Tg mice. Moreover, the lysine lactylation levels of proteins in liver tissues were significantly increased in the HBV-Tg mice. In LX2 cells, we found that pyruvate treatment significantly increased the profibrotic gene expression and lactylation level of PKM2, which promoted its tetramer-to-dimer transition, inhibited its pyruvate kinase activity, and facilitated its nuclear distribution. Through immunoprecipitation, we identified that pyruvate induced PKM2 lactylation at the K206 site. PKM2 knockdown or K206 mutation reduced PKM2 lactylation and abrogated the induction of profibrotic gene expression by pyruvate. Collectively, our findings indicate that HBV infection stimulated pyruvate production, which increased PKM2 lactylation at K206 to promote the expression of profibrogenic genes in HSCs, leading to liver fibrogenesis.

Pathogens doi: 10.3390/pathogens15040430

Authors: Bernardo Almeida Inês Caetano Margarida Santos Ana Duarte Margarida Dias Duarte Sílvia Carla Barros Fábio A. Abade dos Santos Ana Margarida Henriques

Hepatitis E virus (HEV) is a zoonotic pathogen of global concern that circulates in both domestic and wild swine populations. Understanding its presence and dynamics in wildlife reservoirs is crucial for assessing spillover risks and designing One Health surveillance strategies. This study investigated the occurrence, genetic diversity, and evolutionary relationships of HEV in wild boars from mainland Portugal. A total of 120 animals from seven districts were tested, with HEV RNA detected in four cases (3.3%), all from the Évora district near the Spanish border. One positive sample was successfully sequenced, and phylogenetic analysis based on the complete genome classified it within the HEV-3m subtype, clustering with predominantly human-derived sequences from Spain and France, which highlights its zoonotic potential. A second phylogenetic analysis based on a partial genomic fragment, including sequences from domestic pigs in Portugal, revealed the co-circulation of subtypes 3e, 3f, and 3m without clear spatial or temporal patterns. Phylogeographic analysis suggested that the identified strain was most likely introduced from Spain, supporting the hypothesis of cross-border transmission through wild boar movement. No recombination events were detected in the sequence obtained in this study. These findings provide the first molecular evidence of HEV-3m circulation in wild boars in Portugal, offering valuable insight into the HEV strain circulation in European wildlife populations. The zoonotic potential of HEV and the likelihood of interspecies transmission highlight the need for coordinated cross-border surveillance and integrated One Health strategies.

Pathogens doi: 10.3390/pathogens15040429

Authors: Carolina Hurtado-Marcos Fernando Izquierdo Soledad Fenoy Carmen del Águila Jaume Pérez-Griera Salvador Benlloch Cirilo Amorós Carlos García Ballesteros Francisca López Chuliá Juan Carlos Andreu-Ballester Carmen Cuéllar

Crohn’s disease (CD) is frequently accompanied by T-cell lymphopenia and impaired mucosal immunity, conditions that may predispose to intestinal microsporidiosis by Encephalitozoon cuniculi. This prospective case–control study examined the interplay between IL-7/IL-7 receptor (IL-7R) signaling and anti-E. cuniculi immune responses in 50 CD patients and 50 matched healthy controls. Serum IL-7 and anti-E. cuniculi IgG, IgM, IgA and IgE were quantified by ELISA, while intestinal expression of IL-7, CD127 (IL-7Rα) and CD132 (IL-7Rγ) was assessed by RT-PCR. Protein levels of IL-7 and caspase-3 were evaluated by Western blot, and lymphocyte subsets and apoptosis by flow cytometry. CD patients showed reduced anti-E. cuniculi IgG and IgM levels but increased seropositivity, indicating compromised humoral quality despite greater exposure. Compared with controls, CD was associated with decreased serum IL-7, increased mucosal IL-7, downregulated CD132, and diminished caspase-3, suggesting a disrupted IL-7/IL-7R-apoptosis pathway. In CD, IgA- and IgE-skewed responses correlated differentially with caspase-3 and CD56+ γδ T cells, while E. cuniculi seropositivity independently predicted a shorter surgery-free interval. These findings identify a profound dysregulation of the IL-7/IL-7R-CD132-caspase-3 axis in CD and implicate E. cuniculi exposure as a potential marker of impaired mucosal immunity and adverse outcomes.

Pathogens doi: 10.3390/pathogens15040428

Authors: Amanda Garcia Pereira Sarah Raquel Jesus Santos Simões Maitê Cardoso Coelho da Silva Ana Cláudia Calchi Ricardo Bassini-Silva Ana Carolina Castro-Santiago Rosangela Zacarias Machado Marcos Rogério André Karin Werther

Avian trichomonosis is caused by protozoa of the genus Trichomonas, mainly Trichomonas gallinae, which infects the upper digestive tract of birds and is commonly associated with Columbiformes, the main reservoirs of the parasite. This study aimed to investigate the occurrence and genetic diversity of Trichomonas spp. in captive synanthropic birds from southeastern Brazil. Oropharyngeal swabs were collected from 281 birds belonging to 13 avian orders and analyzed using Diamond medium culture, Giemsa-stained smears, and molecular assays. Of the 262 samples submitted to culture analysis, 72 (27.48%) showed trophozoite-like structures under light microscopy. Molecular screening based on the ITS1–5.8S–ITS2 region detected Trichomonas DNA in 76 out of 267 samples with successful DNA extraction (28.46%), including 72 Columba livia domestica from Franca, one Coragyps atratus from Ribeirão Preto, and three rock doves from Jaboticabal. Among the ITS-positive samples, 67 (88.15%) amplified the Fe-hydrogenase gene, and 65 (85.5%) were also positive for the 18S rRNA gene. Only six samples (2.29%) exhibited structures compatible with Trichomonas spp. in Giemsa-stained smears. Phylogenetic analyses based on ITS sequences grouped the isolates into two clades within the Trichomonas gallinae complex. Greater genetic diversity was observed using Fe-hydrogenase and 18S rRNA markers, revealing multiple haplotypes and clades. Molecular assays, particularly PCR applied directly to oropharyngeal swabs, showed higher sensitivity for detecting and characterizing Trichomonas gallinae compared to culture and cytology. These findings highlight the high occurrence and genetic diversity of T. gallinae in captive synanthropic pigeons and reinforce the importance of molecular tools for epidemiological surveillance in wildlife facilities.

Pathogens doi: 10.3390/pathogens15040427

Authors: Meng Xia Yinghui Song Xiaofang Dong Li Gu Yishuo Wang Wen Sun Bin Zhan Yan Yan Limei Zhao

EmCRT is a calreticulin secreted by Echinococcus multilocularis during its infection in host, playing an important role in evading host immune attack as a survival strategy. Our previous study has demonstrated that recombinant EmCRT (rEmCRT) was able to bind to C1q and lectin to interfere with host classical and lectin complement activation pathway, respectively. However, the C1q-binding site on EmCRT and the associated immune evasion mechanism remain unknown. In this study, the C1q-binding site on EmCRT was determined through molecular docking analysis and fragment expression to be localized to the S-domain (EmCRT-S) between Lys140 at the N-domain and Gln292 at the P-domain. The recombinant EmCRT-S protein (rEmCRT-S) was subsequently expressed in bacteria. Functional analysis confirmed that rEmCRT-S was able to bind to human C1q and inhibit C1q-initiated complement activation at the similar level to the full-length rEmCRT, resulting in the reduction in C4b/C3b deposition and antibody-sensitized sheep red blood cell hemolysis. Furthermore, rEmCRT-S binding to C1q suppressed THP-1-derived macrophage chemotaxis and ROS generation. Given that the identified functional domain EmCRT-S provides similar complement regulatory functions to the full-length EmCRT, this domain is a more feasible and practical target for vaccine development against E. multilocularis infection or for inflammatory and autoimmune diseases.

Pathogens doi: 10.3390/pathogens15040426

Authors: Vinícius Machado dos Santos Gabriel da Silva Oliveira Concepta McManus

Sanitization of hatching eggs is part of established poultry management practices, and its effectiveness is essential for productive success. This study aimed to investigate the relationships between microbiological and incubation performance variables obtained from a controlled experimental dataset of hatching eggs subjected to sanitization with essential oils or not under commercial conditions, and to determine the efficacy of these sanitizers, using a multivariate approach. Data were analyzed using principal component, canonical, cluster, and discriminant analysis. The results suggested that bacterial contamination of the eggshell may promote internal contamination, leading to embryonic mortality. Essential oil-based treatments are associated with lower microbial indicators and improved hatchability, while formaldehyde showed an opposite trend despite its antibacterial efficacy. Multivariate analyses clarified the interrelationships between microbiological and incubation performance variables, allowing the identification of response patterns that evidenced the functional efficiency of essential oil–based treatments for hatching egg sanitization under commercial conditions.

Pathogens doi: 10.3390/pathogens15040425

Authors: Refik Bozbuğa Furkan Ulaş Özlem Urtekin Muhammad Aasim Mustafa İmren Rachid Lahlali Muhammad Amjad Ali Fouad Mokrini Abdelfattah Dababat

Climate change significantly impacts agricultural ecosystems through rising temperatures, changing precipitation patterns, increasing atmospheric CO2 levels, and more frequent extreme weather events. These environmental changes have a pronounced effect on plant-parasitic nematodes (PPNs; phylum Nematoda), which cause serious crop losses on a global scale. This review aims to provide a comprehensive evaluation of current knowledge on how major climate change drivers influence the biology, population dynamics, host–plant interactions, and geographic distribution of PPNs in agricultural systems. Recent studies show that rising temperatures accelerate nematode development, increasing the number of generations within a production season and facilitating the spread of many economically important species toward higher latitudes and elevations. Changes in precipitation patterns and soil moisture directly affect nematode survival, mobility, and infection success, and these effects often vary depending on regional conditions and nematode species. Elevated atmospheric CO2 levels modify plant–nematode interactions by increasing root biomass, altering rhizosphere processes, and regulating plant defense pathways (e.g., jasmonic acid and salicylic acid signaling), which may enhance host susceptibility and infection intensity. Furthermore, extreme climate events can disrupt the natural balance in soil ecosystems, weakening natural antagonist–nematode relationships. However, responses of PPNs to climate change are not uniform, and contrasting findings across studies indicate that these responses are strongly shaped by species-specific traits and environmental variability. In addition, future research should focus on long-term and multi-factorial field studies to better capture the combined effects of climate drivers. Overall, climate change is expected to increase PPN prevalence and drive shifts in their geographic distribution, highlighting the need for climate-sensitive and regionally adapted nematode management strategies.

Pathogens doi: 10.3390/pathogens15040424

Authors: Amanda Faier-Pereira Paula Finamore-Araujo Maria Mikaely Ribeiro Brito Alejandro Marcel Hasslocher-Moreno Otacilio C. Moreira

Chagas disease (CD) is a neglected tropical disease caused by the flagellate protozoan Trypanosoma cruzi, representing a major socioeconomic challenge. MicroRNAs (miRNAs) are small non-coding RNA molecules that regulate gene expression, and several pathogens, including T. cruzi, can modulate host miRNA networks. In this context, we hypothesized that host-derived miRNAs could serve as biomarkers in chronic CD. Given the intrinsic lability of RNA, we evaluated the efficacy of a 6 M guanidine-HCl/0.2 M EDTA solution, widely used in the molecular detection of T. cruzi DNA, in preserving mRNAs and miRNAs when mixed in a 1:1 ratio with human blood. Samples with or without guanidine were enriched with exogenous miRNAs (cel-miR-39 and cel-miR-54) and stored at 4 °C. RNase P expression was also evaluated in blood samples stored for up to 120 days and in samples from patients with CD, allowing direct comparison of mRNA stability over time. Samples preserved with guanidine-EDTA showed Ct values that were 4 to 5 cycles lower for all targets analyzed and demonstrated greater RNA stability over time. Taken together, these findings demonstrate that guanidine-EDTA robustly preserves mRNA and miRNAs in human blood, expanding the feasibility of molecular analyses in retrospective samples and corroborating its potential application in the studies of biomarkers of therapeutic response and prognosis in CD.

Pathogens doi: 10.3390/pathogens15040423

Authors: Charles Gotuaco Ang Shreya Eyunni Irwin M. Chaiken

Antiretroviral therapy (ART) effectively controls Human Immunodeficiency Virus Type-1 (HIV-1) infection in people with HIV-1 (PWH), preventing the progression of their infections to AIDS. However, as PWH age, they experience lifestyle- and age-related diseases, notably various types of cancer beyond those traditionally associated with AIDS, with greater incidence and mortality than their non-HIV-1-positive counterparts, despite effective arrest of HIV-1 infection by ART. Dysregulation of redox enzymes presents an underexplored linkage between HIV-1 infection and cancer comorbidity, impacting reactive oxygen/nitrogen species (RONS) management, inflammation, immune function, and mitochondrial function. Chronic HIV-1 infection increases both RONS production and RONS neutralization responses, accelerating development of a sustained RONS-rich environment that still possesses sufficient dampening to prevent outright cytotoxic effects. Such an environment promotes both tumor proliferation and resistance adaptations to chemo- and radiotherapies. This review considers the effects of chronic HIV-1 infection on redox enzyme function and links these effects to tumorigenic mechanisms as potentially shared pathways. We then examine current methods of modulating redox function, consider how these could potentially impact both HIV-1 infection and cancer progression, and lastly propose future methods of co-treatment that could be explored.

Pathogens doi: 10.3390/pathogens15040422

Authors: Samantha P. Stacey Bryan C. Mounce

Polyamines are small, positively charged molecules essential for fundamental cellular processes, including transcription, translation, and membrane fluidity. In the central nervous system (CNS), these molecules serve as homeostatic gatekeepers by modulating neuroreceptors like NMDA and supporting autophagic clearance. While basal polyamine levels are necessary for proper neuronal differentiation and memory formation, their dysregulation is a hallmark of neurodegenerative pathologies such as Alzheimer’s and Parkinson’s diseases. Neurotropic viruses, including poliovirus, Zika virus, and human cytomegalovirus are significant human pathogens that rely on cellular metabolites for their replication, including polyamines. These pathogens exploit polyamines at multiple stages of their life cycles, relying on them for virion stability, cellular attachment, and the stimulation of viral enzyme activity. Notably, diverse viral families share this dependence, making polyamine biosynthesis a prime target for broad-spectrum antiviral therapies. This review covers the current understanding of polyamine metabolism in virus infection and CNS health and disease, as well as considering antiviral therapies targeting host polyamines to limit neurotropic virus infection.

Pathogens doi: 10.3390/pathogens15040421

Authors: Abel Biguezoton Haladou Gagara Chaka Traore Der Dabire Zakaria Bengaly Mahaman Maaouia Abdou Moussa Kader Issoufou Maïmouna Ousmane Marcella Mori Claude Saegerman

Background: Bovine tuberculosis (bTB) caused by Mycobacterium bovis is a major zoonotic disease in West Africa. In Africa, bTB is endemic in cattle with a prevalence ranging from 2% up to 18%. The disease causes significant public health risks due to unpasteurized milk and milk product consumption. In the context of the EU-PRISMA project, which promotes research and innovation for productive, resilient, and healthy agropastoral systems in West Africa, a cross-sectional survey was conducted in dairy herds from Mali and Niger to assess animal, herd, and within-herd bTB prevalence, as well as to identify animal risk factors and predictors of bTB herd status. Method and principal findings: A random cross-sectional survey on dairy cattle farms using comparative intradermal tuberculin test and epidemiological inquiry was performed in four regions of Mali (Bamako, Koulikoro, Mopti, and Sikasso) and three regions of Niger (Tahoua, Dosso, and Tillabéry). Herd and animal prevalence of bTB and within-herd prevalence were significantly higher in Mali (especially in Bamako and Koulikoro) than in Niger. Several risk factors were significantly associated with animals positive to bTB, i.e., the region where animals live, the age range from 3 to 7 years old, and female animals. In addition, in regions with higher bTB prevalence, the herd with slaughtering of animals in the farm and the herd with the presence of an animal assembly area were associated with the most unfavorable status of a herd with regards to bTB. Moreover, the average and the median annual economic losses of bTB at animal level were estimated at €262 and €137 respectively, with large variability depending on the farm (between €46 and €838). Conclusion and significance: This survey provides useful data on bTB epidemiology and economical losses in Mali and Niger and urges for improvement of surveillance systems and prevention and control strategies. Cost-benefit, return of investment, or similar analyses are strongly recommended to help with decision making.

Pathogens doi: 10.3390/pathogens15040420

Authors: Jonghoe Byun

Periodontal disease represents a major global health burden, beginning with gingivitis and progressing to periodontitis, which causes connective tissue breakdown, alveolar bone resorption, and eventual tooth loss. Beyond local pathology, periodontitis is a chronic inflammatory condition with systemic associations, including cardiovascular disease, diabetes, and metabolic disorders. Mesenchymal stem cells (MSCs) and their extracellular vesicles (EVs) have emerged as promising candidates for periodontal regeneration. This review aimed to map the current evidence on MSC-derived EVs (MSC-EVs) in periodontal regeneration, focusing on their mechanisms of action, therapeutic potential, and translational challenges. A comprehensive literature search was conducted across a major biomedical database (PubMed) to identify preclinical and clinical studies investigating MSC-EVs in the context of periodontitis. Data were charted on EV cargo composition, biological functions, regenerative outcomes, and reported limitations. Evidence indicates that MSC-EVs encapsulate bioactive molecules—including antimicrobial peptides, proteins, lipids, and microRNAs—that modulate immune responses, suppress pro-inflammatory signaling, and promote angiogenesis and tissue repair. In periodontal models, MSC-EVs attenuate osteoclast activity, enhance fibroblast proliferation, and stimulate extracellular matrix remodeling, supporting regeneration of periodontal ligament and alveolar bone. Exosome-based approaches demonstrate advantages such as reduced immunogenicity, improved safety, and feasibility for storage and standardization. However, most findings remain preclinical, with limited human data available. To bridge the translational gap, well-designed clinical trials are needed to confirm efficacy and safety while addressing regulatory challenges, GMP standards, and outcome measures. Harnessing their regenerative capacity while mitigating side effects may guide precision-targeted therapies, and continued mechanistic studies with standardized production will be key to advancing MSC-EVs into clinical practice.

Pathogens doi: 10.3390/pathogens15040418

Authors: Adina-Alexandra Nanu Miruna-Ioana Lazăr Dragoș Ștefan Lazăr Corneliu Petru Popescu Maria Nica Simin Aysel Florescu

Background: Early identification of Staphylococcus spp. infective endocarditis (IE) remains clinically challenging but essential for timely initiation of targeted antimicrobial therapy. We aimed to characterize pathogen-specific clinical phenotypes and to develop a pragmatic bedside prediction model for staphylococcal etiology. Methods: We conducted a retrospective cohort study including 112 patients diagnosed with definite IE. Demographic, clinical, echocardiographic, and laboratory data were analyzed. Independent predictors of staphylococcal etiology were identified using multivariable logistic regression, with internal validation by bootstrap resampling. A simplified clinical risk score was derived from regression coefficients. Platelet kinetics were evaluated as a potential complementary biomarker. Results: Patients with staphylococcal IE (n = 66) were younger and exhibited a distinct clinical profile characterized by a high prevalence of intravenous drug use, right-sided valve involvement, and a markedly elevated inflammatory response. Independent predictors included intravenous drug use (OR 7.1, 95% CI 2.45–20.6, p < 0.001), higher C-reactive protein levels (OR 1.08 per unit increase, p = 0.025), and lower oxygen saturation (OR 0.75 per 1% increase, p = 0.007). The model demonstrated good discrimination (AUC 0.82, 95% CI 0.74–0.90) and calibration. The simplified exploratory risk score stratified patients into low-, intermediate-, and high-risk groups, with observed probabilities of 27%, 82%, and 94%, respectively. Conclusions: Staphylococcal IE represents a distinct clinical phenotype. A simple three-variable model enables early bedside identification of high-probability cases and may support risk-adapted management decisions. Nevertheless, external validation is required before clinical implementation of a score.

Pathogens doi: 10.3390/pathogens15040419

Authors: Emma T. Hickman Meagan E. Williams Roberta L. DeBiasi Sarah B. Mulkey

Patients with Lyme disease often face uncertainty when navigating the healthcare system during pregnancy and when seeking healthcare for their children exposed to Lyme disease in utero. Little is known about these families’ experiences. This qualitative study explored the perspectives of 27 parents in the United States who had acute or chronic Lyme-related diagnoses while pregnant. Semi-structured interviews were coded and thematically analyzed. Six themes characterized positive patient–physician interactions: (1) validation of patient experiences, (2) transparency, (3) willingness to learn, (4) shared decision making, (5) empathy, and (6) continuity of care. These findings offer guidance for clinicians counseling patients facing prognostic uncertainty related to Lyme disease and similarly complex conditions.

Pathogens doi: 10.3390/pathogens15040417

Authors: Katarzyna Bartosik Magdalena Świsłocka-Cutter Joanna Werszko Anna Aftyka Klaudia Mária Švirlochová Dana Zubriková Bronislava Víchová Magdalena Raszewska-Famielec Marek Asman

Haematopota pluvialis is a widely distributed hematophagic insect occurring across Eurasia. This horse fly may be a highly efficient mechanical vector of pathogens, including viruses, bacteria, and protozoa. Furthermore, its painful bites can cause local skin lesions and systemic symptoms. The aim of this study was to determine human exposure to H. pluvialis attacks in various types of open space habitats in Eastern Poland and to perform molecular screening of these tabanids for the presence of hematopathogens: Bartonella spp. and Anaplasma phagocytophilum. Specimens of H. pluvialis were collected at three distinct sites in Eastern Poland. The presence of Bartonella spp. and A. phagocytophilum was investigated using PCR-based methods. In total, 141 H. pluvialis females were analyzed. The molecular analysis of the rpoB gene fragment yielded one new haplotype of Bartonella sp. in 0.7% (1) of all studied samples, which may hypothetically exhibit zoonotic potential. Anaplasma phagocytophilum was not detected in the studied material. Moreover, a high level of human and animals exposure to horse fly bites was noted in the studied areas of Eastern Poland. The present results highlight the need for further targeted research on H. pluvialis to quantify pathogen prevalence, transmission efficiencies, and conditions facilitating pathogen transmission in natural settings.

Pathogens doi: 10.3390/pathogens15040416

Authors: Angioletta Lasagna Giacomo Pozza Maddalena Matone Cinzia Fasola Lorenzo Ruggieri Nicla La Verde Paolo Pedrazzoli Davide Dalu

Background: People living with HIV (PLWH) have a substantially increased risk of both AIDS-defining cancers (ADCs) and non-AIDS-defining cancers (NADCs), which remain a major cause of morbidity despite effective antiretroviral therapy (ART); this review aims to integrate current epidemiological, molecular, and clinical evidence on HIV-associated oncogenesis. Methods: A structured literature search was conducted in PubMed (2000–2026) using predefined keywords, including “HIV”, “cancer”, “oncogenesis”, and “immune dysregulation”, with inclusion of original studies, systematic reviews, and meta-analyses meeting predefined quality criteria. Results: Available evidence indicates that HIV contributes to cancer development through both direct and indirect mechanisms: viral proteins such as Tat, Nef, and Vpr disrupt apoptosis, DNA repair, and cell cycle regulation, while chronic immune activation, persistent inflammation, and immunosuppression impair tumor immune surveillance and facilitate oncogenic viral co-infections, including Epstein–Barr virus, human papillomavirus, and human herpesvirus 8. Emerging pathways, such as epigenetic alterations, microRNA dysregulation, metabolic reprogramming, and the contribution of HIV reservoirs to pro-tumorigenic microenvironments, further modulate cancer risk. Conclusions: HIV may function as a cofactor that enhances the effects of oncogenic viruses by promoting viral persistence and immune dysregulation; while biologically plausible, direct evidence linking HIV to amplification of tumorigenesis in humans remains limited.

Pathogens doi: 10.3390/pathogens15040415

Authors: Alessia Vetri Andrea Basso Caterina D’Onofrio Tobia Pretto Edoardo Turolla Federica Marcer Eleonora Fiocchi Giuseppe Arcangeli Luana Cortinovis Ewa Bilska-Zając Vasco Menconi

Haplosporidium edule is a haplosporidian parasite originally described in the common edible cockle (Cerastoderma edule) along the European Atlantic coast. In this study, we report the first detection of H. edule in the olive-green cockle (Cerastoderma glaucum) from the northern Adriatic Sea, representing both a novel host record and a new geographic occurrence. During a cross-sectional study conducted in May 2019, 90 C. glaucum specimens were collected from three lagoon sites in northeastern Italy. Histological examination of soft tissues revealed haplosporidian developmental stages, including plasmodia, sporoblasts and mature spores, within connective tissues of the mantle, digestive gland, gills and between gonadal tubules in eight individuals from the Goro Lagoon. Molecular characterization based on a fragment of the small subunit ribosomal DNA showed high similarity with the previously published H. edule sequence. Host identification was confirmed through cytochrome c oxidase subunit I barcoding together with morphological and histological analyses. These findings indicate that H. edule has a broader host range than previously recognized. Although prevalence was relatively low, the detection of this parasite in a new host species and geographic area highlights the importance of continued surveillance, particularly in the context of climate change, shellfish translocations and the expansion of aquaculture activities.

Pathogens doi: 10.3390/pathogens15040414

Authors: Neha Raut Anis A. Chaudhary Harshad Patil Supriya Shidhaye Ruchi Khobragade Milind Umekar Mohamed A. M. Ali Rashmi Trivedi

Background/Objectives: Antimicrobial resistance (AMR) is a critical global public health challenge driven by inappropriate and excessive antimicrobial use (AMU) across human, animal, and environmental sectors. Method: This narrative review synthesizes recent evidence on antimicrobial utilization and resistance patterns. A structured search of PubMed, Scopus, and Web of Science was conducted for studies published between 2015 and 2025. Eligible sources included surveillance reports, registry-based analyses, and clinical studies. Data were qualitatively analyzed to identify key trends and regional variations. Result: Marked geographical variation in AMR was observed. Carbapenem resistance in Escherichia coli remains low globally (2–3%) but is higher in Southeast Asia (17–18%) and India (~40%). Klebsiella pneumoniae shows consistently high resistance (>40% globally; ~54% in India), while Pseudomonas aeruginosa exhibits stable resistance levels (35–45%). Resistance prevalence increases from primary to tertiary care settings, reflecting greater antimicrobial exposure. Vulnerable populations—including pediatric, elderly, pregnant, and immunocompromised patients—face higher risks of antimicrobial exposure and adverse outcomes, including nephrotoxicity, hepatotoxicity, and microbiome disruption. WHO AWaRe data indicate a global shift toward increased use of Watch-category antibiotics. Stewardship interventions, such as audit and feedback, prescribing restrictions, rapid diagnostics, and decision support systems, effectively reduce inappropriate AMU. Conclusions: Integrated, data-driven antimicrobial stewardship and robust surveillance systems are essential to mitigate the global burden of AMR.

Pathogens doi: 10.3390/pathogens15040413

Authors: Xiao Yang Hui Ma Min Zhu Xinyu Song Jiahao Feng

Background: Pneumonia of unknown etiology (PUE), defined as pneumonia cases without an identified pathogen at the time of clinical presentation, represents a critical clinical warning signal for emerging infectious disease (EID) outbreaks with pandemic potential. Yet, conventional pathogen-centric surveillance systems suffer from an inherent blind spot: they cannot detect early clustering signals before the causative agent is identified, creating a window of vulnerability during novel pathogen emergence. To address this gap, this study aims to develop a deep learning model that leverages unstructured chest imaging text—a routinely available clinical data stream—to enable real-time, automated screening of PUE cases and early warning of EID clusters, independent of prior pathogen knowledge, within an integrated multi-pathogen surveillance framework. Methods: We retrospectively collected data from 8860 patients with respiratory illnesses at a tertiary hospital in Beijing, China, including 980 PUE cases (11.1%) and 7880 known-etiology pneumonia cases. A deep learning model (RoBERTa with attention enhancement) was developed using unstructured chest imaging reports. The Matthews correlation coefficient (MCC) curve was employed to determine the optimal decision threshold. Model performance was assessed for PUE case identification and clustering signal detection on a test set. Results: The model achieved an area under the receiver operating characteristic curve of 0.986 (95% CI: 0.981–0.991). At the optimal threshold of 0.08, selected by maximizing the Matthews correlation coefficient (MCC)—a balanced metric that accounts for all four confusion matrix outcomes—sensitivity was 89.8%, and specificity was 97.0% for identifying PUE cases. In a simulated surveillance exercise, the model showed a high correlation between the predicted and actual case counts (Pearson’s r = 0.901), suggesting its potential to detect abnormal clustering signals prior to pathogen identification. Conclusions: The developed model demonstrates potential to detect clustering signals of PUE caused by unknown pathogens and can be integrated with hospital information systems, providing a feasible, low-cost tool for integrated surveillance of pathogens with pandemic potential. This approach enables earlier outbreak detection and supports public health decision-making during the critical window before pathogen identification.

Pathogens doi: 10.3390/pathogens15040412

Authors: D. Katterine Bonilla-Aldana Jorge Luis Bonilla-Aldana Lysien Zambrano Alfonso J. Rodriguez-Morales

Yellow fever (YF) remains a re-emerging vector-borne zoonotic disease in tropical regions of the Americas despite the availability of an effective vaccine. In South America, the virus is maintained through a jungle transmission cycle involving Haemagogus and Sabethes mosquitoes and non-human primates (NHPs), which act as amplifying hosts and key epidemiological sentinels. This narrative review examines the current status of YF epizootics in South America, with a focus on the role of NHPs in viral circulation, early detection, and spillover risk to human populations. We synthesize recent evidence on epizootic patterns across endemic countries, the differential susceptibility of neotropical primates, and the ecological and environmental drivers influencing transmission, including deforestation, habitat fragmentation, and human encroachment into forested areas. In addition, we analyze current surveillance strategies, including wildlife monitoring, entomological and genomic surveillance, and their integration within a One Health framework. This review highlights that YF epizootics are expanding geographically and are closely linked to environmental change and human–ecosystem interactions. Strengthening integrated, multidisciplinary surveillance systems is essential to improve early detection, guide vaccination strategies, and prevent human outbreaks. These findings underscore the critical importance of operationalizing the One Health approach to enhance preparedness and response to YF in South America.

Pathogens doi: 10.3390/pathogens15040411

Authors: Karel Petrzik Sára Brázdová

Pseudomonas aeruginosa is a widespread pathogen that causes acute and chronic diseases in various organisms, including humans. Treating this antibiotic-resistant bacterium is challenging, so alternative or supplementary treatment strategies are desirable. Six novel bacteriophages specific to P. aeruginosa were isolated and classified into the genera Septimatrevirus, Kochitakasuvirus, Bruynoghevirus, and a new, unnamed genera related to Napahavirus, and Kantovirus. Their genomes were annotated and further characterized. We used the Drosophila melanogaster insect model to predict the efficacy of the phages in terms of their curative function on other organisms. Flies were chronically infected by feeding them bacteria and were subsequently treated with individual bacteriophages. The results of the Kaplan–Meier survival test revealed differences in phage efficacy and supported the hypothesis that the phages had a curative effect. These mentioned phages extended the flies’ lifespan.

Pathogens doi: 10.3390/pathogens15040410

Authors: Jenyffie Araújo Belizário Maria Eduarda Brites Jardine Gabrielle Lameado Pereira Murilo Molina Stefani Ralciane de Paula Menezes Denise von Dolinger de Brito Röder Reginaldo dos Santos Pedroso Sérgio Ricardo Ambrósio Gil Benard Regina Helena Pires

Candida parapsilosis is a major cause of healthcare-associated infections due to its persistence on abiotic surfaces and efficient transmission via healthcare workers’ hands. This study evaluated the antifungal efficacy and safety of clinically relevant antiseptics against 60 C. parapsilosis clinical isolates using a surface carrier test designed to simulate contamination and disinfection events on hospital surfaces. Antifungal activity was assessed by logarithmic reduction (log10) assays on surface carriers and by minimum inhibitory concentration (MIC) testing. Potential synergistic interactions between antiseptics and selected phytochemicals were investigated using checkerboard assays, and toxicity was evaluated in vivo using Caenorhabditis elegans. Surface carrier assays showed that 70% ethanol and 0.5% alcoholic chlorhexidine (CHG) achieved the highest fungicidal activity, with reductions of up to 5 log10 after 1 min exposure at 25 °C. Polyhexamethylene guanidine hydrochloride (PHMGH) displayed consistently low MIC values (0.4–0.9 ppm) and intermediate surface activity. CHG combined with eugenol or menthol produced strong synergistic interactions, reducing CHG MICs from up to 6250 ppm to as low as 20 ppm (>300-fold). Toxicity assays revealed a narrow safety margin for CHG, whereas PHMGH showed a more gradual concentration-dependent toxicity profile. These findings highlight clinically relevant differences in antiseptic performance and identify combination strategies that may reduce CHG exposure while maintaining antifungal efficacy.

Pathogens doi: 10.3390/pathogens15040409

Authors: Janayna Barroso dos Santos Hanna Gabriela da Silva Oliveira André de Medeiros Costa Lins Edson Moleta Colodel Agnes de Souza Lima Henrique dos Anjos Bomjardim Flavio Roberto Chaves da Silva Cíntia Daudt Valeria Dutra Felipe Masiero Salvarani

Pythiosis is a neglected infectious disease caused by the aquatic oomycete Pythium insidiosum and remains underrecognized in cattle, particularly in tropical regions. Here, we report the first molecularly confirmed outbreak of bovine pythiosis in the Amazon biome, affecting more than 400 animals raised under extensive production systems and areas with prolonged exposure to standing water. Clinically affected cattle presented ulcerative and exudative cutaneous lesions, predominantly involving the distal limbs. Given the diagnostic challenges associated with pythiosis, etiological confirmation was achieved through quantitative PCR (qPCR) targeting the internal transcribed spacer (ITS) region of P. insidiosum, providing rapid and specific molecular detection during the outbreak investigation. Therapeutic interventions were implemented as part of routine field management, including intramuscular triamcinolone combined with topical copper sulfate; this regimen was associated with clinical improvement in a substantial proportion of affected animals, though treatment efficacy was not formally evaluated. The outbreak occurred in flood-prone pastures during the rainy season, highlighting the role of aquatic environments in pathogen transmission. These findings expand the current understanding of bovine pythiosis in tropical ecosystems and underscore the importance of molecular diagnostics, outbreak surveillance, and a One Health approach for the identification and management of water-associated pathogens in livestock.

Pathogens doi: 10.3390/pathogens15040408

Authors: Gokhan Aydin Taner Akyol

Background and Aim: Ulcerative colitis (UC) is a chronic inflammatory bowel disease characterized by immune dysregulation. Environmental factors, including infectious agents, have been proposed to influence disease activity in inflammatory bowel disease. Although Borrelia burgdorferi has been shown to exert complex immunomodulatory effects on host immune responses, its seroprevalence and potential association with disease activity in patients with ulcerative colitis have not been systematically investigated. This study aimed to evaluate the seroprevalence of Borrelia burgdorferi IgG antibodies in patients with ulcerative colitis and to assess the relationship between seropositivity and laboratory markers of disease activity. Methods: In this retrospective observational study, 100 patients with ulcerative colitis (59 males, 41 females; mean age 48.5 ± 17 years) who underwent Borrelia burgdorferi IgG serological testing due to musculoskeletal or neurological symptoms suggestive of possible Lyme disease between October 2020 and October 2024 were included. Demographic characteristics, hematological and biochemical parameters, and inflammatory markers were compared between seropositive and seronegative groups. Due to the retrospective design, validated clinical disease activity indices were not consistently available; therefore, disease activity was indirectly assessed using laboratory inflammatory markers. Results: Among patients with ulcerative colitis, 22% were seropositive for Borrelia burgdorferi IgG. Seropositive patients had significantly lower uric acid, alkaline phosphatase, and C-reactive protein levels compared to seronegative patients (p = 0.001, p = 0.023, and p = 0.020, respectively). Free T4 levels were significantly higher in the seropositive group (p = 0.049). In terms of erythrocyte indices, mean corpuscular volume and mean corpuscular hemoglobin were significantly higher, while RDW-CV values were significantly lower in seropositive patients (all p < 0.05). Conclusion:Borrelia burgdorferi IgG seropositivity in patients with ulcerative colitis was associated with lower laboratory markers of systemic inflammation and a more stable hematological profile. Although causality cannot be established, these findings may suggest a potential association between prior Borrelia exposure and a distinct inflammatory phenotype in UC; however, this relationship should be interpreted with caution. Further prospective and mechanistic studies are warranted to clarify the potential immunological interactions between environmental microbial exposure and inflammatory bowel disease activity.

Pathogens doi: 10.3390/pathogens15040407

Authors: Abdul Kabir Asghar Ali Kamboh Muhammad Abubakar Kinkpe Lionel Abdulkareem Mohammed Matar

Foot and mouth disease (FMD) is a highly contagious transboundary viral disease affecting livestock, causing significant economic losses. This sero-epidemiological study investigated FMD distribution and associated risk factors in cattle and buffaloes along the Pakistan–Afghanistan border. A total of 800 serum samples were collected from cattle (n = 610) and buffaloes (n = 190) and tested for antibodies against FMD viral structural proteins (SP) and non-structural proteins (NSP) using ELISA. Overall, 35.25% (282/800) of samples were NSP-positive, indicating natural infection. Serotype-specific analysis showed serotype O as the most prevalent (66.1%), followed by serotype A (50%) and Asia-1 (32%). Cattle exhibited higher FMD prevalence (37%; 95% CI: 33–40) than buffaloes (30%; 95% CI: 23–37). Significant spatial variations in SP and NSP Seroprevalence were observed across different areas. Risk factor analysis identified male sex, young age (1–2 years), crossbred and exotic breeds, summer season, large herd size, smallholders subsistence production systems, poor body condition, and animal movement as factors associated with significantly higher (p < 0.05) FMD circulation. These findings indicate that FMD is highly endemic in the border region and highlight the critical need for government-led mass vaccination campaigns, targeted risk-based surveillance, and stringent movement control to mitigate disease spread. Implementation of such control strategies is essential to safeguard livestock health and protect the regional economy from substantial losses.

Pathogens doi: 10.3390/pathogens15040406

Authors: Heather M. Ward James J. Lunders Chris Ocegueda

West Nile virus (WNV) remains the most frequently reported locally acquired arboviral infection in the United States, yet many small and mid-sized mosquito abatement districts lack the diagnostic capacity and integrated data systems needed for rapid detection and response. The Canyon County Mosquito Abatement District (CCMAD) in southwestern Idaho undertook a multi-year capacity-building effort to expand arbovirus surveillance, standardize mosquito identification and pooling procedures, and implement triplex RT-qPCR testing for WNV, Western equine encephalitis virus (WEEV), and St. Louis encephalitis virus (SLEV). Historical trapping datasets (2021–2025) were consolidated, geospatially harmonized, and grouped into biologically meaningful sampling units to enable multi-year spatial comparisons. Surveillance revealed recurrent WNV activity annually, with peak transmission occurring between epidemiological weeks 31 and 37. The highest WNV activity occurred in 2023 and 2025, with 192 and 92 positive pools, respectively, while no WEEV or SLEV detections were observed. Enhanced laboratory capacity reduced sample-processing times, decreased the reliance on external confirmatory testing, lowered per-pool testing costs, and enabled same-day reporting to operational staff. In 2025, routine gravid trap surveillance detected a single Aedes aegypti, which was identified morphologically and subsequently confirmed by DNA barcoding, prompting targeted follow-up trapping. CCMAD’s integrated approach provides a scalable model for strengthening local surveillance and response capabilities in resource-limited settings.

Pathogens doi: 10.3390/pathogens15040404

Authors: Xiaohan Tong Ruixue Wang Yaxin Liu Malia B. Potts Shondra M. Pruett-Miller Michael A. Whitt Weikuan Gu Kui Li

The autophagy regulator ATG7 helps maintain cellular homeostasis and has been suggested to modulate aspects of antiviral immune responses. In Drosophila, ATG7-dependent autophagy contributes to host resistance to vesicular stomatitis virus (VSV), a negative-strand RNA virus of family Rhabdoviridae that is widely used for studying viral biology and developing vaccines and virotherapy. However, the role of ATG7 in mammalian cells, especially non-immune cell types, remains unclear. Herein, we systematically examined the impact of ATG7 on VSV infection using CRISPR-edited cell lines derived from murine embryonic fibroblast (MEF), HeLa, and Huh7.5 cells, in relation to its effect on the expression of antiviral interferon-stimulated genes (ISGs). We found that ATG7 deficiency blocked basal as well as VSV-induced LC3B lipidation, concomitant with moderate reductions in progeny virus yields, while the reconstitution of ATG7 reversed the phenotypes. Mechanistically, ATG7 did not affect viral entry but rather was associated with moderate upregulation of VSV RNA replication. Intriguingly, ATG7 inhibited baseline ISG expression, and this correlated with its pro-VSV effect in all three cell types, while its suppression of innate immune responses elicited post-VSV infection did not. Altogether, these data provide new insights into the role of ATG7 in regulating VSV replication and innate immunity and have implications for developing VSV-based prophylaxis/therapeutics.

Pathogens doi: 10.3390/pathogens15040405

Authors: Dmitry Baboshko Kirill Elfimov Polina Achigecheva Irina Osipova Grigoriy Vlasov Oleg Rozhkov Boyko Margarita Aleksey Totmenin Aleksandr Agaphonov Natalya Gashnikova

Bovine leukemia virus (BLV) is an oncogenic retrovirus and the etiological agent of enzootic bovine leukosis (EBL), which is spread worldwide. This study presents data on the genetic diversity of BLV in the Novosibirsk region of Russia. ELISA-positive samples were selected from six districts of the Novosibirsk region (Dovolnoye, Barabinsk, Tatarsk, Toguchin, Bolotnoye, and Kochenyovo districts). To assess the diversity of circulating BLV genotypes, samples were collected from settlements and districts that were geographically distant from each other and had no shared pasture lands. In total, 1410 bp fragments encoding the env gene region were obtained from 417 BLV-positive samples. Phylogenetic analysis classified 325 BLV strains (77.9%) as genotype 4 (G4) and 92 strains (22.1%) as genotype 7 (G7). A pairwise identity matrix was constructed for 268 amino acid residues. Pairwise identity of BLV amino acid sequences in the gp51 region ranged from 96.6% to 100% for G4 and from 97.4% to 100% for G7. Multiple alignment of the amino acid sequences identified 74 mutations found in the Russian BLV variants. Through the addition of 417 novel env BLV sequences to GenBank, this study significantly expands the foundational data and knowledge of BLV molecular epidemiology in Russia.

Pathogens doi: 10.3390/pathogens15040402

Authors: Marcos Mancilla Adriana Ojeda Yassef Yuivar Maritza Grandón Sebastián Valderrama Marcela Oyarzún Horst Grothusen Pablo Ibarra Patricio Bustos

The incidence of furunculosis in juvenile Atlantic salmon, Salmo salar, has increased in recent years in Chile, with isolates of Aeromonas salmonicida being the primary cause. However, in some cases, molecular diagnostics failed to identify the etiological agent. We previously demonstrated that a proportion of undiagnosed cases was produced by a new A. salmonicida strain. In those cases where the pathogen remained unidentified, we isolated colonies with an A. salmonicida-like appearance. Subsequent phylogenetic analysis presented in this work grouped those A. salmonicida-like isolates within the Aeromonas piscicola clade. Whole genome sequencing confirmed the taxonomic affiliation, giving additional insights into virulence and antibiotic resistance markers. Indeed, one of the strains showed reduced susceptibility to oxytetracycline. Virulence potential was assessed by in vivo testing in S. salar, which resulted in disease with pathognomonic signs of furunculosis. Although the pathogen presents common antigens with A. salmonicida, the current vaccine triggered only a modest IgM response against A. piscicola in the field. Our results support the hypothesis that the increasing incidence of furunculosis in Chile cannot solely be ascribed to the emergence of the new less-virulent A. salmonicida strain, but may partially result from furunculosis-like infections caused by A. piscicola strains which exhibit a comparable virulence level.

Pathogens doi: 10.3390/pathogens15040403

Authors: Juan Manuel Hernández-Domínguez Roberto González-Garduño Edgar Castro-Saines Rodolfo Lagunes-Quintanilla Roger Iván Rodríguez-Vivas Agustín Olmedo-Juárez Jorge Alberto Cortes-Morales Claudia Yesenia León-González

The objective of this study was to evaluate essential oils (EOs) as an alternative control method for Rhipicephalus microplus and Amblyomma mixtum larvae. The EOs used were obtained by steam distillation from the leaves of cinnamon (Cinnamomum verum), mexican mint (Plectranthus amboinicus), lemongrass (Cymbopogon citratus), peppercorns (Pimenta dioica), and peruvian pepper tree (Schinus molle). To evaluate the acaricidal effect of EOs against the larvae of two tick species, a larval immersion test (LIT) was performed using six concentrations (10 mg/mL, 5 mg/mL, 2.5 mg/mL, 1.87 mg/mL, 1.25 mg/mL, and 0.6 mg/mL), in addition to a negative control group (water + ethanol) and three positive controls (organophosphate, formamidine, and pyrethroid). LIT results were obtained 48 h after exposure. Data were processed using Probit procedure to determine the lethal concentrations at 50% (LC50), 95% (LC95), and 99% (LC99). For R. microplus, 99% mortality was obtained at concentrations as low as 1.4 mg/mL for S. molle, while the highest LC99 was recorded with P. dioica at 23 mg/mL. In the case of A. mixtum, higher concentrations were required to achieve a high mortality rate. EO of P. amboinicus had the lowest acaricidal effect, requiring 26.2 mg/mL to achieve an LC99, while S. molle required a concentration of 6.9 mg/mL to achieve an LC99.

Pathogens doi: 10.3390/pathogens15040401

Authors: Monika Janeczko Elżbieta Kochanowicz

This study evaluated the hemolytic activity of Candida albicans isolates from the female reproductive tract and investigated the in vitro effects of quinalizarin on fungal growth, hemolysis, and ECE1 expression. Ninety-four clinical C. albicans isolates and three ATCC reference strains were analyzed. Hemolytic activity was quantified in culture supernatants and normalized per 107 cells. Antifungal susceptibility and the effect of quinalizarin on hemolysis were assessed using broth microdilution and hemolysis assays. Expression of the ECE1 gene was evaluated by quantitative real-time PCR in three selected hemolytic strains. Drug interactions between quinalizarin and fluconazole were determined using the fractional inhibitory concentration index (FICI). Among the 97 tested strains, 78 exhibited hemolytic activity with variable intensity. Quinalizarin demonstrated antifungal activity, with MIC values ranging from 2 µg/mL to 256 µg/mL, and showed synergistic effects with fluconazole in selected strains. Exposure to quinalizarin at subinhibitory concentrations reduced ECE1 transcript levels to 22.8–73.6% of controls (p < 0.05) in the analyzed strains. However, the phenotypic effect on hemolysis was limited, with residual activity remaining high: 82% (p < 0.05), 93.7% (p < 0.05), and 83% (p < 0.05) relative to untreated controls in C. albicans ATCC 10231, ATCC 90028, and a clinical isolate, respectively. FICI analysis confirmed synergistic interactions between quinalizarin and fluconazole. This preliminary in vitro study highlights the need for further investigation into the relationship between ECE1 expression, candidalysin-mediated damage, and the antifungal potential of quinalizarin.

Pathogens doi: 10.3390/pathogens15040399

Authors: Lakkhana Sadaow Patcharaporn Boonroumkaew Rutchanee Rodpai Oranuch Sanpool Tongjit Thanchomnang Marcello Otake Sato Pewpan M. Intapan Hiroshi Yamasaki Yasuhito Sako Toni Wandra Kadek Swastika Wanchai Maleewong

Background: Human cysticercosis, caused by the larval stage (cysticerci) of the pork tapeworm Taenia solium, is an important zoonotic disease. The disease is prevalent in developing countries where porcine cysticercosis is common and undercooked pork is habitually consumed. Objective: This study aimed to develop an immunochromatography-based test kit for the rapid diagnosis of human cysticercosis using low-molecular-weight antigens purified from cyst fluid of the T. solium Asian genotype to detect specific IgG antibodies in whole blood. The kit was designated as “the cysticercosis whole-blood test kit (iCys WB kit).” Methods: It was evaluated under laboratory conditions using 164 whole-blood samples, of which 21 were from confirmed cysticercosis cases. The results of the iCys WB kit, which detects anti-T. solium (cysticercus) antibodies in simulated whole blood samples, were compared with results from corresponding human serum samples. Results: When using both sample types, iCys WB kit demonstrated an accuracy of 98.8%, a sensitivity of 91.7%, a specificity of 100%, a positive likelihood ratio of 0, a negative likelihood ratio of 0.083, and an ROC area of 0.96. The agreement between results obtained from simulated whole-blood and serum samples showed perfect concordance. Conclusions: The iCys WB kit is a valuable easy-to-handle diagnostic tool and may be applicable for supporting clinical diagnosis at the point of care.

Pathogens doi: 10.3390/pathogens15040400

Authors: Elena G. Olson Emily A. Matiak Joshua A. Jendza Steven C. Ricke

Background: Campylobacter jejuni, a leading foodborne pathogen in poultry, relies heavily on iron for survival and colonizes the gastrointestinal tract (GIT). Iron supplementation in poultry diets can inadvertently promote pathogen growth, particularly when excess or poorly absorbed iron accumulates in the lower GIT. Encapsulated iron products, such as SQM® Iron, offer a controlled-release mechanism that may mitigate this risk by reducing iron availability to microbes. Objective: This study evaluated the effects of free (FeSO4) versus polysaccharide–iron complex (PIC) on C. jejuni growth under iron-limited conditions, hypothesizing that encapsulated iron would support slower and more limited bacterial proliferation due to delayed iron release. Methods: Growth kinetics of C. jejuni ATCC 700819 were assessed in chelated Mueller–Hinton broth supplemented with three iron concentrations (10, 20, and 50 ppm) of FeSO4, PIC, or PIC matrix without iron. Optical density was measured every 20 min over 48 h under microaerophilic conditions. Maximum growth rate (µmax) and carrying capacity (K) were derived using non-linear curve modeling. ANOVA evaluated statistical significance with Tukey’s HSD post hoc comparisons. Results: Free iron (FeSO4) consistently supported the highest µmax and K values across both trials, indicating rapid and robust C. jejuni proliferation. The effect of encapsulated iron was variable: at higher concentrations (50 ppm) it approached FeSO4 performance, but at lower concentrations (10 ppm) its effect differed markedly between trials, sometimes supporting growth comparable to free iron and sometimes supporting substantially slower growth. The PIC matrix alone did not promote growth. These variable results indicate that the relationship between encapsulated iron and C. jejuni proliferation is complex and concentration-dependent. Conclusions: Free iron consistently promotes robust C. jejuni growth due to immediate bioavailability. The impact of encapsulated iron on C. jejuni proliferation is nuanced and variable, particularly at lower concentrations, suggesting its role in pathogen control is not straightforward and requires further investigation under controlled conditions. Furthermore, in vivo research is warranted to validate its utility in poultry pathogen management strategies.

Pathogens doi: 10.3390/pathogens15040398

Authors: Nguyen Khoi Quan Andrew W. Taylor-Robinson

Infections caused by arboviruses, a diverse group of viral pathogens transmitted by biting arthropod vectors, mainly mosquitoes, ticks, and midges, can cause a range of illnesses in humans, from mild, influenza-like symptoms to severe neurological complications including encephalitis and viral hemorrhagic fever. According to 2024 World Health Organization statistics, vector-borne diseases collectively account for over 700,000 human deaths annually, with mosquito-borne infections such as dengue, chikungunya, Zika, and yellow fever constituting a growing and significant proportion of this burden. What was once considered a problem localized to poorly resourced settings in tropical and subtropical regions is now becoming a pervasive global challenge. This is due largely to a combination of factors including climate change, transcontinental travel, and urbanization, with the geographical spread and intensity of arboviral outbreaks reaching unprecedented levels during the current century. In much the same way that the escalating global burden of bacterial infections resistant to antibiotics has been described as a silent pandemic, the insidious rise of arboviruses begs questions regarding outbreak preparedness, prevention and control. Here, we highlight the pressing need for comprehensive strategies that incorporate various health sectors to mitigate the emergence and resurgence of arboviral diseases. Future directives that should be prioritized are outlined. As demonstrated by epidemiological trends and historical outbreak data, an orchestrated global response is critical not only for managing current threats but also for preventing future epidemics.

Pathogens doi: 10.3390/pathogens15040397

Authors: Viona Osei Emmanuel Kuufire Rejoice Nyarku Kingsley E. Bentum Tyric James Asmaa Elrefaey Temesgen Samuel Woubit Abebe

The genus Listeria comprises diverse bacteria with significant public health relevance, particularly Listeria monocytogenes. A comparative genomic analysis of ten representative Listeria species was conducted using 33 high-quality genome assemblies to investigate core and accessory genome dynamics and identify candidate diagnostic loci. Pangenome reconstruction was performed using the Roary Integer Linear Programming Bacterial Annotation Pipeline (RIBAP) to classify core, soft-core, and accessory genes, while average nucleotide identity (ANI) analysis assessed genomic relatedness across thresholds of 60–95%. Functional annotation of core and species-specific genes was conducted using Genome Annotation and Information Analysis (GAIA). Core genes were highly conserved and associated with essential cellular functions, whereas the accessory genome contributed to species-level diversification and ecological adaptation. Candidate molecular markers were derived from accessory genes and evaluated based on presence/absence across genomes, retaining loci present in ≥80% of target strains and absent in non-target strains. Experimental validation of selected primers was performed using two L. monocytogenes reference strains (ATCC 19117 and ATCC BAA-679) with conventional PCR and gel electrophoresis to confirm expected amplicon sizes and specificity. These findings establish a genome-informed, specificity-driven framework for marker development and highlight the accessory genome as a valuable source of diagnostic loci, supporting accurate detection, epidemiological surveillance, and food safety monitoring.

Pathogens doi: 10.3390/pathogens15040396

Authors: Matthew E. Falagas Dimitrios Ragias Dimitrios S. Kontogiannis Laura T. Romanos Paraskevi A. Farazi

The assessment of risk of bias in systematic reviews and meta-analyses is crucial, as it indicates the accuracy of the synthesized and evaluated data and the validity of the presented results and conclusions. Until now, standardized tools for this purpose have been available only for clinical and animal studies, while adapted forms of these tools and novel ones have been proposed for in vitro and laboratory studies. However, none of them have been universally standardized so far. The apparent lack of a risk of bias assessment tool for systematic reviews of in vitro antimicrobial susceptibility testing studies constitutes a methodological flaw in these studies. To this end, we developed a risk of bias assessment tool for in vitro antimicrobial susceptibility testing studies. Our tool assesses the risk of bias across six domains: methodological bias, selection bias, preparation bias (including contamination/cross-contamination bias), measurement/observer bias, reporting and publication bias, and bias related to unreported funding and conflicts of interest. The tool evaluates a total of 16 specific criteria. The risk of bias is graded as low, moderate, or high for each evaluated criterion. The proposed risk of bias assessment tool was tested in a pilot validation study of ten relevant studies by two reviewers independently. We believe that the use of the proposed risk of bias assessment tool will increase the methodological strength of systematic reviews and meta-analyses of in vitro antimicrobial susceptibility testing studies.

Pathogens doi: 10.3390/pathogens15040395

Authors: Viktoria Korff Issam El-Debs Barbara G. Klupp Conrad M. Freuling Jens P. Teifke Thomas C. Mettenleiter Julia Sehl-Ewert

Alphaherpesviruses, including Herpes Simplex Virus 1 (HSV-1) and Pseudorabies Virus (PrV), establish lifelong latency in the nervous system and can cause recurrent disease. While latency has classically been attributed to peripheral sensory ganglia, accumulating evidence indicates that the central nervous system (CNS) may also serve as a site of long-term viral persistence and reactivation. Here, we investigated the CNS as a viral reservoir using the attenuated mutant PrV-∆UL21/US3∆kin, which preferentially targets mesiotemporal brain regions. Following intranasal inoculation, mice were analyzed at 11–14, 21, 28, 42, 105, and 190 days post-infection (dpi). To assess the reactivation potential, a subset of animals received cyclophosphamide/dexamethasone at 170 dpi. Viral transcripts were detected by RNAscope™ in situ hybridization and RT-qPCR targeting the lytic gene UL19 encoding the major capsid protein and the latency-associated transcript (LAT). Histopathology included hematoxylin and eosin staining and immunohistochemistry for CD3, Iba1, GFAP, cleaved caspase-3 and viral glycoprotein gB. UL19 RNA signals displayed marked regional and temporal heterogeneity, with prominent detection in mesiotemporal structures. In contrast, LAT RNA levels remained low overall, with a transient peak during the acute phase. RT-qPCR confirmed high UL19 and LAT transcript levels during early infection, while LAT transcription returned to baseline levels thereafter. Histopathology showed a transition from acute necrotizing meningoencephalitis to prolonged low-grade inflammation with glial activation and focal apoptosis. Notably, UL19 RNA signals strongly correlated with T-cell infiltration, particularly at 42 dpi. Together, these findings define regional and temporal patterns of long-term PrV transcriptional activity and associated neuropathology in the murine CNS.

Pathogens doi: 10.3390/pathogens15040394

Authors: Dylan M. Johnson Sharon Jan Ethan Dunn Jason E. Comer Robert W. Cross Thomas W. Geisbert

Lujo virus (LUJV) is an arenavirus that causes Lujo Hemorrhagic Fever (LHF), a viral hemorrhagic fever that emerged in a 2007 outbreak in Zambia and South Africa with an 80% case fatality rate and evidence of human-to-human nosocomial transmission. There are no approved medical countermeasures for LHF, although several screens have identified lead antiviral compounds. The lack of accessible animal models limits the development of lead compounds and characterization of broadly protective anti-arenavirus compounds such as ribavirin for the treatment of LHF. Here, we present preliminary data characterizing the partially lethal disease caused by LUVJ in STAT-1 deficient mice. Several key hematological, clinical chemistry, and histologic findings common to LHF disease are recapitulated in this model. This work suggests that further characterization of LUJV infection in STAT-1 deficient mice may allow development of a model that would be instrumental in the development of medical countermeasures for LHF.

Pathogens doi: 10.3390/pathogens15040393

Authors: Mollie McDermott Shamim Sarkar Janice O’Brien Karen Gruszynski Barbara Shock Vina Faulkner Lauren Wisnieski

Lyme disease is the most common vector-borne disease in North America. Predicting Lyme disease incidence is a key component of public health preparedness. Previously, we demonstrated that the volume of data searches on Google Trends for terms related to Lyme disease, such as “Lyme” and “tick bite”, can be used as a tool to predict monthly human Lyme disease incidence at the state level. The objective of this project was to build upon our previous work by adding environmental and canine data to our predictive models for the prediction of state-level human and canine Lyme disease incidence. Human data were acquired from state health departments. Canine data were acquired from IDEXX Laboratories. We hypothesized that incorporating a One Health approach with human, animal, and environmental data would improve the predictive ability of the models. The One Health model performed significantly better (Mean Absolute Error [MAE] = 12.1) in predicting human disease incidence in 6 out of 16 states compared to the environmental data model (MAE = 16.5), human search terms model (MAE = 21.4), canine data (search terms + case count) model (MAE = 31.1), and the canine case data model (MAE = 32.0). For canine Lyme disease incidence, the One Health model performed worse (MAE = 330.5) compared to the canine search data model (MAE = 282.3), the human data (search terms + cases) model (MAE = 248.4), and the environmental data (MAE = 221.5) model. However, even the best-performing models had large prediction errors, which limit practical utility. Future studies should incorporate alternative data streams, such as electronic health records and insurance claims, to test predictive ability.