Pathogens

18 pages, 10832 KB

Open AccessArticle

LINC1467 Activates the IPO8–p65 Axis to Restrict Hand, Foot, and Mouth Disease Virus Replication

by Xiaokui Zhang, Jinwei Li, Li Ding, Jihong Zhang, Fan Yang, Yonghan Luo and Wei Chen

Pathogens 2025, 14(10), 1071; https://doi.org/10.3390/pathogens14101071 - 21 Oct 2025

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Hand–foot–mouth disease (HFMD), primarily caused by human enteroviruses (EVs), poses a public health challenge, particularly among infants, due to a lack of effective therapies. Elucidating the molecular interplay between EVs and the host immune response is crucial for developing antiviral treatments. Recent studies [...] Read more.

Hand–foot–mouth disease (HFMD), primarily caused by human enteroviruses (EVs), poses a public health challenge, particularly among infants, due to a lack of effective therapies. Elucidating the molecular interplay between EVs and the host immune response is crucial for developing antiviral treatments. Recent studies have highlighted the significance of long non-coding RNAs (lncRNAs) in regulating host–pathogen interactions; however, the mechanisms of lncRNAs in EV infection remain poorly unexplored. Here, we identified a highly inducible nuclear lncRNA, LINC1467, that is upregulated in response to HFMD-related EV infection. Functional analyses revealed that LINC1467 suppresses viral replication. Mechanistically, LINC1467 interacts with nuclear import receptor Importin 8 (IPO8) to form the LINC1467/IPO8/p65 complex, facilitating the phosphorylation and nuclear translocation of p65, thus promoting the expression of pro-inflammatory cytokines and activating the NF-κB pathway. The antiviral function of LINC1467 was further validated in a mouse model of viral infection. These findings uncover a novel lncRNA-mediated regulatory mechanism in the innate immune response and highlight LINC1467 as a promising target for future antiviral strategies against HFMD-related EVs. Full article

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14 pages, 1991 KB

Open AccessArticle

Influence of Incubation Time for Viability Assessment of Ascaris suum Eggs—Attempts to Optimise the Parasitological Examination

by Radosław Badziak, Jolanta Zdybel, Zbigniew Osiński, Ewa Bilska-Zając, Jacek Karamon, Jacek Sroka, Marta Skubida, Angelina Wójcik-Fatla and Tomasz Cencek

Pathogens 2025, 14(10), 1070; https://doi.org/10.3390/pathogens14101070 - 21 Oct 2025

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Abstract

The aim of this study was to determine the optimal incubation time for accurate assessment of Ascaris suum eggs viability, an important step in improving parasitological diagnostics. The experiment used A. suum eggs collected from three sources: adult roundworms uteri (U), pig faeces [...] Read more.

The aim of this study was to determine the optimal incubation time for accurate assessment of Ascaris suum eggs viability, an important step in improving parasitological diagnostics. The experiment used A. suum eggs collected from three sources: adult roundworms uteri (U), pig faeces (F) and sewage sludge (S), then incubated at 27 °C and monitored weekly. The number of Petri dishes used for eggs observations for each source was 12 (100 eggs were observed on each Petri dish). Eggs were classified as dead (with clear deformations), viable (with motile larvae) or of uncertain viability (retaining structural integrity but undeveloped). The results showed that eggs from group U had the highest viability (96%) and developed larvae within 3 weeks. In contrast, group F (52% viability) and S (3% viability) showed delayed development, requiring up to 8–12 weeks for a conclusive viability assessment. The differences in the percentage of QE, LE, and DEwere generally statistically significant, except in the case of eggs from sewage sludge in the fourth week of incubation. These results indicate significant differences in egg viability depending on the sample source and emphasise the need for longer incubation times, particularly for environmental samples such as sewage sludge. The study also highlights the limitations of single time point assessments based solely on egg structure, which can lead to misclassification. In conclusion, prolonged incubation improves diagnostic accuracy by allowing a clearer distinction between viable and non-viable eggs, especially in samples with initially uncertain viability. Full article

(This article belongs to the Section Parasitic Pathogens)

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10 pages, 2134 KB

Open AccessArticle

Effects of a Synthetic Isoquinoline Derivative Against Ichthyophthirius multifiliis In Vivo and In Vitro in Grass Carp (Ctenopharyngodon idella)

by Xianqi Peng, Xialian Bu, Weisong Ma, Jinbiao Jiao, Xiaohong Huang, Yu Zhao, Jian Zhu, Lei Huang, Jing Chen, A’qin Zheng, Huantao Qu and Jiayun Yao

Pathogens 2025, 14(10), 1069; https://doi.org/10.3390/pathogens14101069 - 21 Oct 2025

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Abstract

White spot disease, caused by the parasitic Ichthyophthirius multifiliis, induces mortality exceeding 80% in intensive aquaculture systems, resulting in global annual losses of >$1 billion. Current chemical controls (e.g., formaldehyde) face environmental persistence and drug resistance challenges. This study developed a synthetic [...] Read more.

White spot disease, caused by the parasitic Ichthyophthirius multifiliis, induces mortality exceeding 80% in intensive aquaculture systems, resulting in global annual losses of >$1 billion. Current chemical controls (e.g., formaldehyde) face environmental persistence and drug resistance challenges. This study developed a synthetic isoquinoline derivative, BHTCA (s)-2-tert-butoxycarbonyl-7-hydroxy-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid), as a biodegradable alternative against I. multifiliis in grass carp (Ctenopharyngodon idella). In vitro assays revealed stage-selective efficacy: theronts exhibited higher susceptibility (EC₅₀ = 0.10 mg/L at 4 h) than tomonts (EC₅₀ = 0.40 mg/L at 24 h), with BHTCA disrupting ciliary structures and compromising cyst integrity. In vivo trials (0.6 mg/L) reduced parasite loads by 78.1% and host mortality by 66.7% versus untreated controls. Acute toxicity tests confirmed a high therapeutic index (LC₅₀ = 16.75 mg/L; >167× effective concentration). With demonstrated efficacy, low production cost, and minimal eco-toxicity, BHTCA presents a sustainable strategy for Ichthyophthiriasis management in aquaculture. Full article

(This article belongs to the Special Issue Fish Pathogens: An Ongoing Challenge for Aquaculture)

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17 pages, 2369 KB

Open AccessArticle

Genome-Wide Identification of Novel miRNAs and Infection-Related Proteins in Leishmania major via Comparative Analysis of the Protozoa, Vectors, and Mammalian Hosts

by Tianyi Liu, Jinyang Qian, Yicheng Yan, Xi Zeng and Zhiyuan Yang

Pathogens 2025, 14(10), 1068; https://doi.org/10.3390/pathogens14101068 - 21 Oct 2025

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Abstract

Leishmania major is a unicellular protozoan that causes cutaneous leishmaniasis in mammals and is mainly transmitted by the sand fly Phlebotomus papatasi. However, the contribution of microRNAs (miRNAs) and protein-coding genes to its pathogenic mechanisms remains largely unexplored. In this study, we [...] Read more.

Leishmania major is a unicellular protozoan that causes cutaneous leishmaniasis in mammals and is mainly transmitted by the sand fly Phlebotomus papatasi. However, the contribution of microRNAs (miRNAs) and protein-coding genes to its pathogenic mechanisms remains largely unexplored. In this study, we systematically analyzed miRNAs and protein-coding genes in L. major, its insect vector, and mammalian hosts. Comparative genomic analysis revealed 2963 conserved proteins shared among the three groups, highlighting a core set of proteins across protozoa, vectors, and hosts. Among mammals, human proteins exhibited the highest homology with L. major, while P. papatasi displayed the lowest proportion of homologs. Functional annotation of 94 hypothetical proteins identified 27 infection-related proteins, including 24 protein kinases and three tyrosine phosphatases, which may represent novel therapeutic targets. In addition, an EST-based approach identified 29 novel miRNAs in L. major. Phylogenetic analysis indicated that these miRNAs diverged into two distinct evolutionary branches, and homology analysis revealed that seven miRNAs were absent in all mammalian species. For example, miR-10117-3p was detected only in nematode Heligosmoides polygyrus. Furthermore, miRNA-gene interaction network analysis highlighted four key genes potentially involved in L. major infection. Collectively, our findings expand current knowledge of protozoan virulence by identifying novel miRNAs and infection-related proteins and provide promising candidates for future drug development against leishmaniasis. Full article

(This article belongs to the Section Parasitic Pathogens)

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14 pages, 7377 KB

Open AccessCase Report

Pulmonary Tuberculosis in the 19th Century: A Historical Case Study of Dr. Șerban Eminovici, Romanian Physician and Brother of Poet Mihai Eminescu

by Andrei Ionut Cucu, Catalin M. Buzduga, Navena Widulin, Alexandru Nemtoi, Amelian Madalin Bobu, Claudia Florida Costea, Roxana Filip, Vlad Porumb, Anca Petruta Morosan, Alexandru Carauleanu, Anca Sava, Elena Porumb-Andrese, Emilia Patrascanu, Camelia Tamas and Andreas G. Nerlich

Pathogens 2025, 14(10), 1067; https://doi.org/10.3390/pathogens14101067 - 21 Oct 2025

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Abstract

Background: In the 19th century, pulmonary tuberculosis was the leading cause of death in Europe, responsible for up to one-quarter of all mortality. Before Robert Koch’s discovery of the tubercle bacillus in 1882 and the advent of effective therapies, treatment relied on rest, [...] Read more.

Background: In the 19th century, pulmonary tuberculosis was the leading cause of death in Europe, responsible for up to one-quarter of all mortality. Before Robert Koch’s discovery of the tubercle bacillus in 1882 and the advent of effective therapies, treatment relied on rest, high-caloric diets, and sanatoria. Objectives: This study aims to reconstruct the medical biography of Dr. Șerban Eminovici (1841–1874), Romanian physician and elder brother of poet Mihai Eminescu, and to contextualize his life and death within the broader history of tuberculosis and pre-antibiotic medical practice. Methods: We conducted a historical case study using archival sources, including university registers from Erlangen, Munich, and Vienna, hospital admission records from the Charité Hospital in Berlin, and contemporaneous correspondence. Secondary literature on the history of tuberculosis and the Eminovici family was also reviewed. Results: Eminovici pursued medical studies across Central Europe, obtaining his doctorate in Vienna and later practicing medicine in Berlin, where he was a member of the Berliner Medizinische Gesellschaft. Despite early signs of respiratory illness, treated at spa resorts such as Gleichenberg, his condition progressed to advanced pulmonary tuberculosis with neuropsychiatric complications. Hospital records confirm his admission to the Charité on 10 October 1874, and his death from “Lungenschwindsucht” (pulmonary tuberculosis) on 29 November 1874, at age 33. His trajectory illustrates both the transnational mobility of Romanian intellectual elites and the therapeutic limitations of pre-antibiotic medicine. Conclusions: The case of Dr. Șerban Eminovici highlights the devastating impact of tuberculosis on 19th-century intellectuals, the reliance on lifestyle-based therapies before the discovery of the tubercle bacillus, and the importance of Central European medical networks in shaping Romanian professional identities. Beyond its biographical significance, this case underscores the persistent social and cultural burden of tuberculosis in Eastern Europe. Full article

(This article belongs to the Section Bacterial Pathogens)

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17 pages, 6022 KB

Open AccessArticle

Evaluation of Suitable Reference Gene During the Development of Paired or Unpaired Female Schistosoma japonicum on the 18th and the 23rd Days Post Infection

by Suwen Wang, Liang Feng and Jun Sun

Pathogens 2025, 14(10), 1066; https://doi.org/10.3390/pathogens14101066 - 21 Oct 2025

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Abstract

Background: Identifying optimal housekeeping genes is essential to accurately quantify gene expression dynamics across the 18th day (male and female begin to pair) and the 23rd day (female begin to sex mature) post infection of Schistosoma japonicum, because this process involves selecting [...] Read more.

Background: Identifying optimal housekeeping genes is essential to accurately quantify gene expression dynamics across the 18th day (male and female begin to pair) and the 23rd day (female begin to sex mature) post infection of Schistosoma japonicum, because this process involves selecting suitable housekeeping genes to ensure the reliability and accuracy of all subsequent expression analyses, thereby improving the precision of biological interpretations. Schistosoma japonicum transcriptomics reveals marked stage-dependent variation in candidate reference genes, which directly challenges the long-standing hypothesis that commonly recommended reference genes remain stably expressed throughout the 18th day and the 23rd day post-infection developmental phases and therefore emphasizes the critical need for careful selection and rigorous validation in any specific experimental context. Methods: In this study, seven widely reported genes (GAPDH, TUBA, ACTB, SOD1, TP, ND and PS) of Schistosoma japonicum were systematically validated by combining Solexa high-throughput sequence analysis with targeted qPCR experiments to identify the most suitable reference genes on the 18th day and the 23rd day post infection of Schistosoma japonicum, and the expression stability of these seven candidate genes was then comprehensively evaluated using four complementary algorithms—the ΔCT method and the GeNorm V3.5, BestKeeper, and NormFinder software applications. Results: GAPDH displayed the most consistent expression profiles, whereas TUBA exhibited the least stability, particularly at the specific time points of 18 and 23 days post infection in both paired and unpaired female Schistosoma japonicum. Conclusions: The suitability of any housekeeping gene is strongly dependent on the study’s specific context and experimental conditions. Therefore, the conclusions drawn here are explicitly limited to the developmental window of 18 and 23 days post infection. Rigorous, stage-specific validation is indispensable before reliable quantitative gene expression analyses can be performed. Full article

(This article belongs to the Section Parasitic Pathogens)

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20 pages, 3464 KB

Open AccessArticle

Specificity of Primers and Probes for Molecular Diagnosis of Leishmania (Leishmania) chagasi in Dogs and Wild Animals

by Giovanna Zandonadi Haber, Leila Dias da Costa, Erick Bruno Monteiro Costa, Railton Farias Araújo, Tainá Negreiros de Souza, Luciana do Rêgo Lima Queiroz, Bruno Tardelli Nunes Diniz, Edivaldo Costa Sousa Junior, Lívia Carício Martis, Patrícia Karla Santos Ramos and Fernando Tobias Silveira

Pathogens 2025, 14(10), 1065; https://doi.org/10.3390/pathogens14101065 - 21 Oct 2025

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Abstract

Molecular tools, especially real-time polymerase chain reaction (qPCR), are relevant tools for laboratory diagnosis due to their sensitivity, specificity, rapid results, and ability to quantify parasite load. This study evaluated the specificity of the LEISH-1/LEISH-2 primer pair with the TaqMan MGB probe in [...] Read more.

Molecular tools, especially real-time polymerase chain reaction (qPCR), are relevant tools for laboratory diagnosis due to their sensitivity, specificity, rapid results, and ability to quantify parasite load. This study evaluated the specificity of the LEISH-1/LEISH-2 primer pair with the TaqMan MGB probe in serum samples previously classified by indirect Enzyme-Linked Immunosorbent Assay (ELISA) (30 positive dogs, 30 negative dogs; 9 positive wild animals and 16 negative wild animals) using in silico analyses (Primer-BLAST, Multiple Alignment using Fast Fourier Transform-MAFFT^®, Geneious, RNAfold, and SnapGene) and Real-Time Polymerase Chain Reaction (qPCR) experimentation. Unexpected amplification occurred in all negative samples, revealing critical specificity failures mainly associated with the probe. In silico analyses confirmed these findings, indicating structural incompatibilities and low selectivity of the sequences. To address this limitation, a new set of oligonucleotides, named GIO, was designed. Computational analyses showed superior performance of GIO, with greater structural stability, absence of unfavorable secondary structures, and improved specificity. Although experimental validation is still required, the results suggest that GIO has strong potential for use in more robust and reliable diagnostic protocols for visceral leishmaniasis across different epidemiological contexts. Full article

(This article belongs to the Section Parasitic Pathogens)

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12 pages, 845 KB

Open AccessArticle

Risk Factors and Clinical Outcomes of Invasive Fungal Infections in Patients with Severe COVID-19: A Case–Control Study

by Nosheen Nasir, Syed Abbas Moazzam Kazmi, Joveria Farooqi, Muhammad Irfan and Kauser Jabeen

Pathogens 2025, 14(10), 1064; https://doi.org/10.3390/pathogens14101064 - 21 Oct 2025

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Abstract

Background: Invasive fungal infections (IFIs) in patients with COVID-19 contribute to significant morbidity and mortality, with reported incidence between 5% and 26.7%. COVID-19-associated pulmonary aspergillosis (CAPA), candidiasis (CAC), mucormycosis (CAM), and Pneumocystis jirovecii pneumonia (PJP) are the most common IFIs in this population. [...] Read more.

Background: Invasive fungal infections (IFIs) in patients with COVID-19 contribute to significant morbidity and mortality, with reported incidence between 5% and 26.7%. COVID-19-associated pulmonary aspergillosis (CAPA), candidiasis (CAC), mucormycosis (CAM), and Pneumocystis jirovecii pneumonia (PJP) are the most common IFIs in this population. Methodology: We conducted a case–control study in the ratio of 1:2 between March 2020 and April 2022 using institutional COVID-19 registry data. The cases were severe COVID-19 patients with IFIs, and the controls were severe COVID-19 patients without IFIs. Multivariate logistic regression was used to identify independent risk factors, with adjusted odds ratios (aOR) and 95% confidence intervals (CIs). The outcomes for the study were to assess the clinical outcomes, i.e., in-hospital mortality and length of hospitalization in a subgroup of severe COVID-19 patients who had IFIs. A p-value < 0.05 was considered significant. Results: Among 5368 COVID-19 patients admitted to hospital during the study period, 1333 had a severe infection. Of these, 158/1333 (11.8%) met the criteria for IFIs, with a median age of 65 years and 71% male predominance. Diabetes (53.8%) and hypertension (57.6%) were the most common comorbid conditions. Acute respiratory distress syndrome (ARDS) developed in 55% of patients. Overall mortality was 48%. For the case control analysis, 119 patients with IFIs were selected as cases and 240 patients without IFIs were selected as controls. On univariate analysis ARDS was significantly associated with IFIs (OR: 1.91; 95% CI: 1.23–2.99, p-value = 0.004). Patients with IFIs had higher odds of being on hemodialysis compared to those without IFIs (OR: 2.17; 95% CI: 1.18–3.99; p-value = 0.013). Mechanical ventilation was found to be independently associated with IFIs in multivariate logistic regression analysis (OR: 2.5, 95% CI: 1.58–3.96, p-value < 0.001). The odds for in-hospital death in patients with IFIs were 2.19 (95% CI: 1.35–3.56; p-value < 0.001) compared to patients without IFIs. The median hospital stay for patients with IFIs was markedly longer (14 days) compared to 8 days in patients without IFIs. Conclusions: IFIs significantly worsened outcomes in severe COVID-19 patients, leading to increased mortality and prolonged hospital stays. Mechanical ventilation was an independent risk factor for IFIs. Full article

(This article belongs to the Section Fungal Pathogens)

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14 pages, 1441 KB

Open AccessArticle

Morphological and Molecular Studies of Tetracotyle-Type Metacercariae of the Genus Cotylurus Szidat, 1928 (Trematoda) from the Gravel Snail Lithoglyphus naticoides (Gastropoda) and Host Sex Dependent Differences in Infection Rate

by Gražina Stanevičiūtė, Virmantas Stunžėnas and Romualda Petkevičiūtė

Pathogens 2025, 14(10), 1063; https://doi.org/10.3390/pathogens14101063 - 20 Oct 2025

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Abstract

The Ponto-Caspian invader, the gravel snail Lithoglyphus naticoides (C. Pfeiffer, 1828), is infected with a diverse community of digenetic trematodes in its colonized range and most often serves as first intermediate host. We have performed the parasitological examination of L. naticoides snails sampled [...] Read more.

The Ponto-Caspian invader, the gravel snail Lithoglyphus naticoides (C. Pfeiffer, 1828), is infected with a diverse community of digenetic trematodes in its colonized range and most often serves as first intermediate host. We have performed the parasitological examination of L. naticoides snails sampled in Kaunas water reservoir (Lithuania) and found yet unknown strigeid metacercariae of the tetracotyle type using these snails as second intermediate host. In this study, we report data on morphology and molecular analysis based on two markers, the partial 28S rDNA gene and the ITS2 region of these metacercariae. Based on the comparative molecular and phylogenetic analysis, the metacercaria detected in L. naticoides was identified as Cotylurus cornutus (Rudolphi 1809) Szidat, 1928. Differences in metacercariae infection between snail sexes were assessed. The prevalence of infection in L. naticoides was significantly higher in males than in females. Additional molecular markers of tetracotyle of C. cornutus from Stagnicola palustris (O. F. Müller, 1774) and furcocercaria of Cotylurus strigeoides Dubois, 1958 from Physa fontinalis (Linnaeus, 1758), sampled in Curonian Lagoon, Lithuania, were obtained for the first time and used for comparative phylogenetic analysis. Full article

(This article belongs to the Special Issue Parasitic Helminths and Control Strategies)

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15 pages, 530 KB

Open AccessSystematic Review

Resistance of Acinetobacter baumannii Complex Clinical Isolates to Sulbactam–Durlobactam: A Systematic Review of Data from In Vitro Studies

by Matthew E. Falagas, Laura T. Romanos, Dimitrios Ragias and Charalampos Filippou

Pathogens 2025, 14(10), 1062; https://doi.org/10.3390/pathogens14101062 - 20 Oct 2025

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Abstract

Introduction: Due to the limited therapeutic options for patients with Acinetobacter baumannii complex infections, a new combination antimicrobial agent, sulbactam–durlobactam, has been developed. In this systematic review, we evaluated the available data on the resistance of A. baumannii complex clinical isolates to sulbactam–durlobactam. [...] Read more.

Introduction: Due to the limited therapeutic options for patients with Acinetobacter baumannii complex infections, a new combination antimicrobial agent, sulbactam–durlobactam, has been developed. In this systematic review, we evaluated the available data on the resistance of A. baumannii complex clinical isolates to sulbactam–durlobactam. Methods: We performed a thorough search of four databases for relevant studies. The Clinical and Laboratory Standards Institute (CLSI) sulbactam–durlobactam breakpoint for A. baumannii complex susceptibility was used (MIC value ≤4 mg/L). Data on the presence of genes of various β-lactamases were also analyzed. Results: From 182 identified articles, 84 were thoroughly screened. Data extraction was performed on 20 articles (published 2017–2025) reporting on a total of 10,412 A. baumannii complex clinical isolates. Among the various β-lactamases genes present, the OXA subvariants OXA-23/OXA-23-like were the most common (in 561 isolates). The proportions of non-selected (consecutive) A. baumannii isolates found to be resistant to sulbactam–durlobactam were 1.2%, 1.2%, and 4.6% in the three studies, and with non-susceptibility (resistance and intermediate resistance) were 2%, 2.1%, and 4.6% in three other studies. Non-susceptibility was very rare among A. calcoaceticus, A. nosocomialis, and A. pittii isolates (0%, 0.3%, and 0.6%, respectively). The proportion of carbapenem-resistant A. baumannii isolates with resistance was 0–5.2%. The proportion of A. baumannii isolates selected for their reduced susceptibility profile (including reduced susceptibility to cefiderocol) with resistance was 1.4–27.3%. Discussion: The low proportion of sulbactam–durlobactam resistance among A. baumannii complex isolates supports the consideration of the use of this new antibiotic for its approved indications. Full article

(This article belongs to the Special Issue Bacterial Resistance and Novel Therapeutic Approaches)

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17 pages, 3312 KB

Open AccessArticle

Fungus Among Us: An 8-Year Retrospective Study of Fungal Infections at a Tertiary-Care Hospital in Bucharest, Romania

by Alina Maria Borcan, Mihaela-Cristina Olariu, Teodora Gabriela Anghel, Elena Rotaru, Bianca Secuiu, Madalina Simoiu, Narcis Copca, Dragos Cretoiu and Laura Georgiana Caravia

Pathogens 2025, 14(10), 1061; https://doi.org/10.3390/pathogens14101061 - 20 Oct 2025

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Abstract

Despite the devastating consequences of fungal disease, research struggles to catch up to present needs. This study aims to give a broad perspective on the situation, investigating patterns and distribution of fungal pathogens and monitoring trends of resistance to antifungal drugs, over an [...] Read more.

Despite the devastating consequences of fungal disease, research struggles to catch up to present needs. This study aims to give a broad perspective on the situation, investigating patterns and distribution of fungal pathogens and monitoring trends of resistance to antifungal drugs, over an 8-year timeframe, at the National Institute of Infectious Diseases “Prof. Dr. Matei Balș” in Bucharest, Romania. Samples were inoculated on Sabouraud or Brilliance Candida Agar media; strains were identified using MALDI-TOF MS; and antifungal sensitivity testing was performed using E-Tests strips, VITEK2 Compact and MICRONAUT-AM automatic systems. Candida albicans, accounting for 42% of the positive samples, was the most common pathogen observed, with only 17% of the isolates being susceptible to all antifungals tested, while it was also predominant and deadly in the ICU. The emerging Candidozyma auris, found in 8% of the candidoses, exhibited a fluconazole resistance rate of 96.3%. Of the Aspergillus fumigatus strains, 35.7% showed resistance to azoles, and 25% to amphotericin B. In the ICU, more than half of A. flavus-, A. fumigatus- or A. niger-related cases culminated in death. Antifungal resistance is not to be treated lightly, as it is still a complex and dynamic threat, with devastating consequences. Full article

(This article belongs to the Section Fungal Pathogens)

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19 pages, 10016 KB

Open AccessArticle

A Novel Enterococcus Phage Endolysin Lys22 with a Wide Host Range Against Mixed Biofilm of Enterococcus faecalis, Staphylococcus aureus, and Acinetobacter baumannii

by Ziqin Yang, Xue Du, Nannan Hu, Meng-Ai Feng, Jiaoyang Xu, Hailin Jiang, Na Zhang, Honglan Huang, Jinghua Li and Hongyan Shi

Pathogens 2025, 14(10), 1060; https://doi.org/10.3390/pathogens14101060 - 20 Oct 2025

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Abstract

The global surge in multidrug-resistant (MDR) bacterial pathogens has created an urgent imperative for innovative antimicrobial strategies. Enterococcus faecalis, Staphylococcus aureus, and Acinetobacter baumannii demonstrate remarkable antibiotic resistance and dominate hospital-acquired infections. These bacteria often form biofilms, a complex community structure [...] Read more.

The global surge in multidrug-resistant (MDR) bacterial pathogens has created an urgent imperative for innovative antimicrobial strategies. Enterococcus faecalis, Staphylococcus aureus, and Acinetobacter baumannii demonstrate remarkable antibiotic resistance and dominate hospital-acquired infections. These bacteria often form biofilms, a complex community structure that shields them from immune system phagocytosis, resists antibiotic penetration, and enhances their survival in hostile environments. In clinical cases, these bacteria often form mixed biofilms and lead to treatment failures. Phages and their derivatives have emerged as promising candidates in the fight against drug-resistant bacteria. Lys22, an endolysin derived from an enterococcus phage, has been cloned and demonstrated to possess a broad host range, effectively targeting E. faecalis, various Staphylococcus species, and A. baumannii. When applied to the biofilms formed by these bacteria, Lys22 was found to significantly inhibit both simple and complex biofilms in vitro. Virulent genes, including agrA, sarA, and icaA in S. aureus; asa1, cylA, and gelE in E. faecalis; and OmpA and lpsB in A. baumannii were also downregulated by Lys22. Notably, Lys22 also exhibited a robust protective effect against dual or triple infections involving E. faecalis, S. aureus, and A. baumannii in a zebrafish embryos model, highlighting its potential as a therapeutic agent in combatting multi-bacterial infections. Full article

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

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28 pages, 2452 KB

Open AccessArticle

Beyond Microbiological Analysis: The Essential Role of Risk Assessment in Travel-Associated Legionnaires’ Disease Outbreak Investigations

by Antonios Papadakis, Eleftherios Koufakis, Vasileios Nakoulas, Leonidas Kourentis, Theodore Manouras, Areti Kokkinomagoula, Artemis Ntoula, Maria Malliarou, Kyriazis Gerakoudis, Katerina Tsilipounidaki, Dimosthenis Chochlakis and Anna Psaroulaki

Pathogens 2025, 14(10), 1059; https://doi.org/10.3390/pathogens14101059 - 20 Oct 2025

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Abstract

Between April and May 2025, an outbreak of travel-associated Legionnaires’ disease (TALD) occurred, involving six cases at a hotel in Crete, Greece. Including two cases reported in 2023 and two additional cases from 2016 to 2017, ten cases were associated with this accommodation [...] Read more.

Between April and May 2025, an outbreak of travel-associated Legionnaires’ disease (TALD) occurred, involving six cases at a hotel in Crete, Greece. Including two cases reported in 2023 and two additional cases from 2016 to 2017, ten cases were associated with this accommodation site. All TALD cases were reported by the European Legionnaires’ Disease Surveillance Network (ELDSNet). In compliance with the European Centre for Disease Prevention and Control (ECDC) surveillance and investigation protocols for hotels associated with the patient’s stay, local public health authorities conducted on-site inspections at the hotel by collecting water samples and performing risk assessments, while simultaneously recording the required epidemiological, environmental, and physicochemical data. A total of 181 statistically analyzed water samples showed positive rates for L. pneumophila of 12.71% (95% CI: 7.86–17.56) for (≥50 CFU/L) and 6.08% (95% CI: 2.60–9.56) for (≥1000 CFU/L). Risk assessments identified 18 stagnation points, systemic maintenance deficiencies, and high cumulative structural (30/52) and water (36/71) system risk scores. Low microbiological positivity of water samples does not necessarily equate to low risk, thus necessitating continuous risk assessment, implementation of Water Safety Plans (WSPs), and integrated monitoring by accommodation facilities to prevent LD cases. Full article

(This article belongs to the Section Bacterial Pathogens)

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1 pages, 165 KB

Open AccessCorrection

Correction: Simião et al. Involvement of Inflammatory Cytokines, Renal NaPi-IIa Cotransporter, and TRAIL Induced-Apoptosis in Experimental Malaria-Associated Acute Kidney Injury. Pathogens 2024, 13, 376

by Gustavo Martins Simião, Kleber Simônio Parreira, Sandra Gabriela Klein, Flávia Batista Ferreira, Fernanda de Souza Freitas, Eduardo Ferreira da Silva, Neide Maria Silva, Murilo Vieira da Silva and Wânia Rezende Lima

Pathogens 2025, 14(10), 1058; https://doi.org/10.3390/pathogens14101058 - 20 Oct 2025

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Abstract

In the original publication [...] Full article

19 pages, 1221 KB

Open AccessArticle

Evaluation of Gastrointestinal Endoparasites in Cattle in Central Spain: Focus on Calicophoron daubneyi with Coprological, Epidemiological, and Anthelmintic Insights

by Nélida Fernández Pato, Óscar García Barrero, Elvira Rodríguez Velasco, Félix Valcárcel Sancho and Jaime Galán Elvira

Pathogens 2025, 14(10), 1057; https://doi.org/10.3390/pathogens14101057 - 19 Oct 2025

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Abstract

Calicophoron daubneyi, a rumen fluke increasingly reported in European livestock, has emerged as a relevant parasitic threat in cattle. This study investigated the prevalence and seasonal dynamics of gastrointestinal endoparasites in 382 fecal samples from 40 beef cattle farms (26 extensive and [...] Read more.

Calicophoron daubneyi, a rumen fluke increasingly reported in European livestock, has emerged as a relevant parasitic threat in cattle. This study investigated the prevalence and seasonal dynamics of gastrointestinal endoparasites in 382 fecal samples from 40 beef cattle farms (26 extensive and 14 semi-extensive) in central Spain. Samples were analyzed using flotation, sedimentation, and modified McMaster techniques, complemented by PCR confirmation of trematodes and a 25-variable epidemiological survey. C. daubneyi was detected in 38.74% of samples and 77.5% of farms, surpassing Fasciola hepatica (13.09%), gastrointestinal nematodes (42.15%), and Eimeria spp. (16.75%). Mixed infections were frequent. Seasonal shedding patterns varied by parasite, with C. daubneyi peaking in spring and winter. Statistical analyses (Kruskal–Wallis, ANOVA, Mann–Whitney U) revealed significant seasonal differences and confirmed higher F. hepatica egg counts in extensive systems (p = 0.0012). Anthelmintic treatment was infrequent and mainly guided by coprological diagnosis; ivermectin, closantel, albendazole, and nitroxinil were the most used drugs, though none fully effective against C. daubneyi. Anthelmintic resistance was not evaluated in this study. These findings confirm the emergence of C. daubneyi in central Spain and highlight the need for targeted surveillance and seasonally adjusted control strategies. Full article

(This article belongs to the Special Issue Emerging and Re-Emerging Parasitic Diseases)

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14 pages, 633 KB

Open AccessArticle

Comparative Efficacy of Horse and Chicken Serum for the In Vitro Cultivation of Mycoplasma hyorhinis Clinical Isolates

by Yi-Chia Li, Yu-Wei Tseng, Wei-Hao Lin, Chao-Nan Lin and Ming-Tang Chiou

Pathogens 2025, 14(10), 1056; https://doi.org/10.3390/pathogens14101056 - 19 Oct 2025

Viewed by 333

Abstract

Mycoplasma hyorhinis is an important respiratory pathogen in swine, yet optimal culture conditions for high-yield propagation remain undefined. This study compared horse serum (HS) and chicken serum (CS) at graded concentrations (10%, 20%, 30%) for their ability to support in vitro growth of [...] Read more.

Mycoplasma hyorhinis is an important respiratory pathogen in swine, yet optimal culture conditions for high-yield propagation remain undefined. This study compared horse serum (HS) and chicken serum (CS) at graded concentrations (10%, 20%, 30%) for their ability to support in vitro growth of four clinical M. hyorhinis isolates (strains A, B, C, and D). Cultures were prepared in modified Friis medium, and growth performance was assessed by final titer (color changing unit, CCU/mL) and time-to-detection at 10² and 10⁴ CCU/mL. All media supported growth, but HS consistently outperformed CS in both yield and growth kinetics. The highest titers (10⁹ CCU/mL) and shortest detection times (3.6–6 days) were observed in 20% HS for most strains. Increasing HS concentration to 30% reduced yield for several strains, suggesting a concentration-dependent inhibitory effect. CS demonstrated limited but strain-dependent growth support, with comparable performance to HS for strain B at lower thresholds. These findings identify 20% HS as an optimal supplement for efficient M. hyorhinis cultivation, while highlighting the potential of CS as a cost-effective alternative under certain conditions, with implications for diagnostic reagent production and vaccine development. Full article

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14 pages, 1928 KB

Open AccessArticle

Prevalence of Rotavirus in Diarrheic Piglets on RVA-Vaccinated and Non-Vaccinated Farms

by Weronika Rybkowska, Aleksandra Woźniak, Nicole Bakkegård Goecke, Lars Erik Larsen, Piotr Cybulski and Tomasz Stadejek

Pathogens 2025, 14(10), 1055; https://doi.org/10.3390/pathogens14101055 - 18 Oct 2025

Viewed by 408

Abstract

Rotaviruses (RVs) are an important cause of piglet diarrhea. This study aimed to determine the prevalence of rotavirus A, B, and C (RVA, RVB and RVC) in two RVA-vaccinated (VAC) and four non-vaccinated (NON-VAC) farms, and the impact of RVA vaccination on production [...] Read more.

Rotaviruses (RVs) are an important cause of piglet diarrhea. This study aimed to determine the prevalence of rotavirus A, B, and C (RVA, RVB and RVC) in two RVA-vaccinated (VAC) and four non-vaccinated (NON-VAC) farms, and the impact of RVA vaccination on production parameters. Additionally, RVs prevalence in consecutive weekly groups from one vaccinated and one non-vaccinated farm was assessed. Diarrheic feces or ileum content were screened for RVs using real-time RT-PCR. In VAC, no RVA or RVB was detected, while RVC was found in all the samples (15/15). In NON-VAC, RVA, RVB, and RVC were detected in 10.5%, 13.2%, and 52.6% of samples, respectively. RVC was the most prevalent species in longitudinal study, while RVA was found in single samples. RVB was detected in one sample from the vaccinated farm, and in four out of five groups from the non-vaccinated farm. The pre-wean mortality and weaning weight were lower in the vaccinated than in the non-vaccinated farm. Low RVA prevalence and no noticeable improvement in weaning outcomes suggest vaccination was probably unjustified. Our study emphasizes the importance of comprehensive screening before and after vaccination and highlights the importance of including RVB and RVC in diagnostics of neonatal diarrhea. Full article

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17 pages, 2606 KB

Open AccessReview

Plasmid Genomic Dynamics and One Health: Drivers of Antibiotic Resistance and Pathogenicity

by Célia P. F. Domingues, João S. Rebelo, Francisco Dionisio and Teresa Nogueira

Pathogens 2025, 14(10), 1054; https://doi.org/10.3390/pathogens14101054 - 18 Oct 2025

Viewed by 536

Abstract

Seen through a One Health perspective, plasmids act as global links, connecting human, animal, and environmental microbiomes while broadening the ecological scope of resistance and virulence. By combining knowledge about plasmid classification, mobility, resistance, virulence, and data sources, this review emphasizes their key [...] Read more.

Seen through a One Health perspective, plasmids act as global links, connecting human, animal, and environmental microbiomes while broadening the ecological scope of resistance and virulence. By combining knowledge about plasmid classification, mobility, resistance, virulence, and data sources, this review emphasizes their key role as drivers of bacterial evolution and worldwide health risks. Recognizing plasmids as connectors across microbiomes highlights both the urgency and opportunity to address plasmid-mediated resistance with integrated strategies. Current plasmid databases, such as NCBI RefSeq, PLSDB, IMG/PR, and PlasmidScope, have already greatly advanced our understanding of these connections, and they are likely to profoundly alter how we see plasmid biology and One Health relationships. Full article

(This article belongs to the Special Issue Bacterial Pathogenesis and Antibiotic Resistance)

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14 pages, 1586 KB

Open AccessArticle

Real-Time Quaking-Induced Conversion Assay Applied to the Italian Chronic Wasting Disease Monitoring Plan: Comparison of Classical and Innovative Diagnostic Methods

by Maria Mazza, Alessandra Favole, Valentina Campia, Barbara Iulini, Romolo Nonno, Ciriaco Ligios, Davide Pintus, Simone Peletto, Cristina Casalone, Cristiano Corona, Elena Bozzetta and Pier Luigi Acutis

Pathogens 2025, 14(10), 1053; https://doi.org/10.3390/pathogens14101053 - 18 Oct 2025

Viewed by 440

Abstract

CWD surveillance and diagnosis are important issues in Europe since its detection in Norway, as some of its strains, like that of classical scrapie, are contagious. In addition, there are concerns as several matters about CWD are not yet known. Although diagnostic methods [...] Read more.

CWD surveillance and diagnosis are important issues in Europe since its detection in Norway, as some of its strains, like that of classical scrapie, are contagious. In addition, there are concerns as several matters about CWD are not yet known. Although diagnostic methods for the active surveillance in bovine and small ruminants have been able to detect the European CWD strains, a retrospective study on Italian wild red deer (Cervus elaphus) samples was performed to compare the results obtained from rapid screening tests, authorized according to EU Regulation 999/2001, and the RT-QuIC, a highly sensitive method in the detection of prion disease infection. A total of one hundred brainstems and medial retropharyngeal lymph nodes were selected out of those received from the CWD Italian surveillance system. Confirmed CWD-positive and -negative samples were included in the study as controls. All of the samples were first tested with the HerdChek BSE–Scrapie Antigen Test and then using the RT-QuIC. The rapid test was negative in all brainstem and lymph node samples. RT-QuIC analyses showed only one red deer brainstem sample positive for seeding activity, while all lymph nodes were negative, including the one from this case. This positive brainstem sample was then re-extracted and retested using two different recombinant prion protein substrates (Ha90-231; BV23-231) and their different batches from the first analyses. Seeding activity was consistently confirmed across both substrates and extractions, with positive signals detected down to dilutions of 10⁻⁴ using rPrP Ha90-231 and as low as 10⁻⁶ with rPrP BV23-231. The additional diagnostic investigations performed on this red deer using the alternative rapid test (TeSeE SAP Combi), Western blot, and immunohistochemistry showed negative results both in the brainstem and lymph nodes. This study showed that overall, the results obtained with the HerdChek BSE–Scrapie Antigen Test and RT-QuIC agree except in one case. Our findings highlight the potential of the RT-QuIC method to detect very low levels of PrP^Sc-associated seeding activity that may escape detection using classical methods. While seeding activity does not always equate to infectivity, only a bioassay will confirm the real disease status of this Italian case. These findings support the integration of RT-QuIC as a powerful complementary tool within existing surveillance frameworks to strengthen early detection and diagnostic accuracy. Full article

(This article belongs to the Collection Prions and Chronic Wasting Diseases)

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21 pages, 2543 KB

Open AccessArticle

Multi-Marker Approach for the Identification of Different Heterodera Species (Nematoda: Heteroderidae)

by Maria João Camacho, Maria L. Inácio and Eugénia de Andrade

Pathogens 2025, 14(10), 1052; https://doi.org/10.3390/pathogens14101052 - 18 Oct 2025

Viewed by 301

Abstract

Cyst nematodes of the genus Heterodera are important plant-parasitic nematodes that cause significant crop losses worldwide but are often overlooked due to their non-specific symptoms and complex biology. This study assessed Heterodera diversity in Portugal using an integrative molecular approach based on four [...] Read more.

Cyst nematodes of the genus Heterodera are important plant-parasitic nematodes that cause significant crop losses worldwide but are often overlooked due to their non-specific symptoms and complex biology. This study assessed Heterodera diversity in Portugal using an integrative molecular approach based on four genetic markers (mtCOI, 18S rDNA, ITS, and 28S rDNA). Five valid species were identified: Heterodera cruciferae, H. mani, H. schachtii, H. trifolii, and H. zeae, with H. mani reported for the first time in the country. A distinct taxon from Coimbra (central Portugal) may represent a new or unsequenced species, highlighting gaps in reference datasets. Among the markers, mtCOI was the most effective, though some taxa remained unresolved. These results reinforce the value of multi-marker approaches, contribute with new sequences, and improve diagnostic capability for nematode management. Full article

(This article belongs to the Section Parasitic Pathogens)

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15 pages, 912 KB

Open AccessArticle

Comparative Evaluation of Organic and Commercial Treatments Against Varroa destructor in Apis mellifera: Implications for Honey Yield in Northeastern Mexico

by Jesús Humberto Reyna-Fuentes, Oscar Vicente Vazquez-Mendoza, Mirelly Venecia Mireles-Villanueva, Daniel López-Aguirre, Juana Maria Coronado-Blanco, Ruben Alberto Muñoz-Sánchez and Francisco Reyes-Zepeda

Pathogens 2025, 14(10), 1051; https://doi.org/10.3390/pathogens14101051 - 18 Oct 2025

Viewed by 314

Abstract

Infestation by Varroa destructor represents one of the major challenges for beekeeping, as it compromises both colony health and honey productivity. The objective of this study was to evaluate the efficacy of different organic treatments for the control of V. destructor and their [...] Read more.

Infestation by Varroa destructor represents one of the major challenges for beekeeping, as it compromises both colony health and honey productivity. The objective of this study was to evaluate the efficacy of different organic treatments for the control of V. destructor and their effect on honey production in Apis mellifera colonies located on the central region of Tamaulipas, Mexico. A total of 150 colonies were assigned to five treatments: T1, oxalic acid with glycerin; T2, sublimated oxalic acid; T3, Thymol, T4; HappyVarr; and T5, an untreated control. Mite infestation (initial and final) and honey production were analyzed using a non-parametric approach and were evaluated with the Kruskal–Wallis test, and when significant differences were detected, Steel–Dwass multiple comparisons were performed. To examine the relationship between infestation reduction and honey yield, Spearman’s rank correlation was applied. No significant differences were observed in the initial infestation levels. However, final infestation levels showed highly significant differences among treatments (p < 0.0001), with T1, T2, T3, and sublimated oxalic acid (T4) significantly reducing mite infestation compared with the control. Sublimated oxalic acid represents the most effective and productive control method under the tested conditions. Honey production also differed significantly among treatments (p < 0.0001), with the highest yields recorded in T3 and T4. A strong negative correlation was detected between final infestation and honey production (p < 0.0001). In conclusion, treatments based on oxalic acid (particularly sublimated) and HappyVarr proved effective in reducing V. destructor infestation and improving honey production, highlighting their relevance as viable alternatives for sanitary management in beekeeping. Full article

(This article belongs to the Special Issue Surveillance, Detection and Control of Infectious Diseases of Bees)

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16 pages, 947 KB

Open AccessArticle

Comparative Analysis of Concentration and Quantification Methods for Antibiotic Resistance Genes and Their Phage-Mediated Dissemination in Treated Wastewater and Biosolids

by Irene Falcó, Ana Allende, Francesca Cutrupi, Rosa Aznar, Gloria Sánchez and Pilar Truchado

Pathogens 2025, 14(10), 1050; https://doi.org/10.3390/pathogens14101050 - 18 Oct 2025

Viewed by 426

Abstract

Antimicrobial resistance poses a growing threat to public health, and integrated surveillance strategies across environmental compartments such as treated wastewater and biosolids can substantially improve monitoring efforts. A key challenge is the diversity of available protocols, which complicates comparability for the concentration and [...] Read more.

Antimicrobial resistance poses a growing threat to public health, and integrated surveillance strategies across environmental compartments such as treated wastewater and biosolids can substantially improve monitoring efforts. A key challenge is the diversity of available protocols, which complicates comparability for the concentration and detection of antibiotic resistance genes (ARGs), particularly in complex matrices. In this study, we compared two commonly used concentration methods—filtration–centrifugation (FC) and aluminum-based precipitation (AP)—and two detection techniques, quantitative PCR (qPCR) and droplet digital PCR (ddPCR), for the quantification of four clinically relevant ARGs: tet(A), bla_CTX-M group 1, qnrB, and catI. Analyses were performed in both secondary treated wastewater and biosolid samples, including their purified bacteriophage-associated DNA fractions. Results showed that the AP method provided higher ARG concentrations than FC, particularly in wastewater samples. ddPCR demonstrated greater sensitivity than qPCR in wastewater, whereas in biosolids, both methods performed similarly, although ddPCR yielded weaker detection. Importantly, ARGs were detected in the phage fraction of both matrices, with ddPCR generally offering higher detection levels. These results provide comparative insights into established methodologies and highlight the value of selecting appropriate protocols based on matrix characteristics and surveillance objectives. Full article

(This article belongs to the Special Issue Antimicrobial Resistance in Foodborne Pathogens: Prevalence, Mechanisms, and Interventions)

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Graphical abstract

9 pages, 1015 KB

Open AccessArticle

Efficacy and Safety of Voriconazole for Difficult-to-Treat Distal Lateral Subungual Onychomycosis (DLSO)

by Aditya K. Gupta, Mesbah Talukder, Elizabeth A. Cooper, Lee Magal and Avner Shemer

Pathogens 2025, 14(10), 1049; https://doi.org/10.3390/pathogens14101049 - 17 Oct 2025

Viewed by 454

Abstract

This retrospective case series evaluated off-label voriconazole for distal lateral subungual onychomycosis (DLSO) unresponsive to standard therapy. Twenty-nine culture-confirmed patients who had failed terbinafine (250 mg daily × 12 weeks) and itraconazole pulses (200 mg twice daily for 1 week/month × 3) received [...] Read more.

This retrospective case series evaluated off-label voriconazole for distal lateral subungual onychomycosis (DLSO) unresponsive to standard therapy. Twenty-nine culture-confirmed patients who had failed terbinafine (250 mg daily × 12 weeks) and itraconazole pulses (200 mg twice daily for 1 week/month × 3) received voriconazole (200 mg twice on day 1, then 200 mg daily for 3–4 months). Assessments occurred at 2, 4, 6–9, and 12 months; the primary endpoint was combined clinical cure (≥90% nail clearance) plus mycological cure (negative KOH and culture) at 12 months. Intention-to-treat included 29 patients; per-protocol included 27 (two did not complete follow-up). In the per-protocol cohort, combined cure was 55.6% (15/27) and mycological cure 74.1% (20/27). Complete clinical cure occurred in 66.7% (18/27); 25.9% (7/27) improved markedly, 3.7% (1/27) mildly, and 3.7% (1/27) showed no improvement. Voriconazole was well tolerated and may be considered for DLSO refractory to terbinafine ± itraconazole. Antifungal stewardship remains essential. Full article

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18 pages, 5522 KB

Open AccessArticle

Campylobacter hepaticus Transcriptomics Identified Genes Involved in Spotty Liver Disease (SLD) Pathogenesis

by Varsha Bommineni, Lekshmi K. Edison, Chaitanya Gottapu, Gary D. Butcher and Subhashinie Kariyawasam

Pathogens 2025, 14(10), 1048; https://doi.org/10.3390/pathogens14101048 - 17 Oct 2025

Viewed by 498

Abstract

Campylobacter hepaticus is the etiological agent of Spotty Liver Disease (SLD), a newly emerging bacterial disease of laying hens resulting in significant mortality and production losses primarily in free-range systems. Although its economic impact continues to grow, the molecular basis of C. hepaticus [...] Read more.

Campylobacter hepaticus is the etiological agent of Spotty Liver Disease (SLD), a newly emerging bacterial disease of laying hens resulting in significant mortality and production losses primarily in free-range systems. Although its economic impact continues to grow, the molecular basis of C. hepaticus pathogenesis remains poorly understood. In this study, we conducted transcriptomic profiling of C. hepaticus in three host-relevant conditions, exposure to chicken bile, infection of a chicken liver hepatocellular carcinoma (LMH) cell line, and isolation from liver lesions of naturally infected chickens. Through RNA-seq analysis, we found unique gene expression signatures in each environment. In the bile, C. hepaticus exhibited differential expression of 412 genes, with upregulation of genes related to motility, cell envelope remodeling, glycosylation, nitrate respiration, and multidrug efflux systems, indicating a stress-adaptive, metabolically active lifestyle. In LMH, on the other hand, 125 genes were differentially expressed, primarily reflecting downregulation of motility, oxidative stress response, chaperones, and core metabolic processes, suggesting that these cells adopt a less active, intracellular dormant lifestyle. Transcriptomic analysis of C. hepaticus isolated from the liver identified 26 differentially expressed genes, featuring selective upregulation of genes associated with nitrate respiration, sulfur metabolism, and pyridoxal 5’ phosphate homeostasis, alongside downregulation of the major outer membrane porin (momp), stress response chaperones (dnaK, groL), and genes involved in oxidative stress defense and energy production. Furthermore, the immune evasion-related gene cmeA and a glycosyltransferase gene were found to be highly upregulated. This study presents the first in-depth transcriptomic exploration of C. hepaticus in multiple host relevant niches. Our findings reveal niche-specific gene expression profiles and highlight metabolic and structural adaptations that enable C. hepaticus to survive during bile exposure, persist within host cells, and contribute to liver pathology. These insights provide a basis for identifying novel virulence determinants and may inform the development of targeted interventions, including vaccines or antimicrobial therapy, to control SLD in commercial poultry operations. Full article

(This article belongs to the Section Immunological Responses and Immune Defense Mechanisms)

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55 pages, 3869 KB

Open AccessReview

A Review of the Biology of Chikungunya Virus Highlighting the Development of Current Novel Therapeutic and Prevention Approaches

by Geovana Martelossi-Cebinelli, Jessica A. Carneiro, Kelly M. Yaekashi, Mariana M. Bertozzi, Beatriz H. S. Bianchini, Fernanda S. Rasquel-Oliveira, Camila Zanluca, Claudia N. Duarte dos Santos, Rachel Arredondo, Tiffani A. Blackburn, Rubia Casagrande and Waldiceu A. Verri

Pathogens 2025, 14(10), 1047; https://doi.org/10.3390/pathogens14101047 - 16 Oct 2025

Viewed by 1629

Abstract

Chikungunya virus (CHIKV) is an arthritogenic alphavirus transmitted primarily via Aedes aegypti and Aedes albopictus mosquitoes. Since its identification, CHIKV remained confined to parts of Africa and Asia until the early 2000s, when it expanded to other continents, causing epidemics. Structurally, it is [...] Read more.

Chikungunya virus (CHIKV) is an arthritogenic alphavirus transmitted primarily via Aedes aegypti and Aedes albopictus mosquitoes. Since its identification, CHIKV remained confined to parts of Africa and Asia until the early 2000s, when it expanded to other continents, causing epidemics. Structurally, it is an enveloped virus with a positive-single-stranded RNA genome, which encodes four non-structural proteins (nsP1-nsP4), responsible for viral replication, and five structural proteins (C, E3, E2, 6K, and E1), which form the capsid and envelope. Of these proteins, glycoproteins E1 and E2 are essential for cell recognition and membrane fusion, determining infectivity and viral tropism. CHIKV replication occurs in the cytosol of different cell types, triggering an intense inflammatory and immune response, which manifests clinically as Chikungunya fever (CHIKF). Despite its epidemiological impact, current treatment is limited to symptomatic approaches, including the use of analgesics and anti-inflammatories, as no specific antiviral therapies are available. In response, promising advances are being made, including the development of vaccines, targeted antivirals, and immunotherapies. This article aims to review the main aspects of viral biology, epidemiology, and immunopathogenesis of CHIKV infection, in addition to discussing the main advances in the development of new therapeutic approaches for its control. Full article

(This article belongs to the Special Issue Virus–Host Cell Interactions and Research of New Antivirals)

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20 pages, 4163 KB

Open AccessArticle

From Patterns to Projections: A Spatiotemporal Distribution of Drug-Resistant Tuberculosis in Paraná, Brazil (2012–2023)

by Laiz Mangini Cicchelero, Gustavo Cezar Wagner Leandro, Luciano de Andrade, Jean Eduardo Meneguello, Katiany Rizzieri Caleffi-Ferracioli, Rosilene Fressatti Cardoso and Regiane Bertin de Lima Scodro

Pathogens 2025, 14(10), 1046; https://doi.org/10.3390/pathogens14101046 - 16 Oct 2025

Viewed by 489

Abstract

Drug-resistant tuberculosis represents a challenge with high potential for spread. This ecological study aimed to describe the prevalence and incidence of drug-resistant tuberculosis and analyze its spatial, temporal, and spatiotemporal patterns in Paraná, Brazil, 2012–2023, and forecast trends through 2030. National surveillance data [...] Read more.

Drug-resistant tuberculosis represents a challenge with high potential for spread. This ecological study aimed to describe the prevalence and incidence of drug-resistant tuberculosis and analyze its spatial, temporal, and spatiotemporal patterns in Paraná, Brazil, 2012–2023, and forecast trends through 2030. National surveillance data were analyzed using descriptive statistics, Mann–Kendall trend tests, Global and Local Moran’s I, Kulldorff’s spatial scan statistic, and Seasonal AutoRegressive Integrated Moving Average modeling. A total of 576 cases were identified, corresponding to an incidence of 5.08 per 100,000 inhabitants, with an increasing trend (p < 0.001). After peaking in 2019, incidence declined during the pandemic and rose in 2023. Isoniazid monoresistance was the most frequent profile. Prevalence was higher among males, young adults (15–34 years), and Asian and Black individuals. Spatial distribution showed expansion over time, with early circulation in the West. The North and Northwest exhibited an initial high incidence. Spatial and spatiotemporal analyses identified persistent high-risk clusters in these regions (p < 0.05). Forecasting suggests that, if current conditions persist, the incidence may continue to rise through 2030. The findings highlight the need for surveillance to ensure treatment adherence and interrupt transmission of resistant bacilli, supporting progress toward the global goal of tuberculosis elimination. Full article

(This article belongs to the Section Bacterial Pathogens)

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25 pages, 1246 KB

Open AccessReview

Mitochondrial Dysfunction in Aging, HIV, and Long COVID: Mechanisms and Therapeutic Opportunities

by María Victoria Delpino and Jorge Quarleri

Pathogens 2025, 14(10), 1045; https://doi.org/10.3390/pathogens14101045 - 16 Oct 2025

Viewed by 3285

Abstract

We hypothesize that a unified mitochondrial perspective on aging, HIV, and long COVID reveals shared pathogenic mechanisms and specific therapeutic vulnerabilities that are overlooked when these conditions are treated independently. Mitochondrial dysfunction is increasingly recognized as a common factor driving aging, HIV, and [...] Read more.

We hypothesize that a unified mitochondrial perspective on aging, HIV, and long COVID reveals shared pathogenic mechanisms and specific therapeutic vulnerabilities that are overlooked when these conditions are treated independently. Mitochondrial dysfunction is increasingly recognized as a common factor driving aging, HIV, and long COVID. Shared mechanisms—including oxidative stress, impaired mitophagy and dynamics, mtDNA damage, and metabolic reprogramming—contribute to ongoing energy failure and chronic inflammation. Recent advancements highlight new therapeutic strategies such as mitochondrial transfer, transplantation, and genome-level correction of mtDNA variants, with early preclinical and clinical studies providing proof-of-concept. This review summarizes current evidence on mitochondrial changes across aging and post-viral syndromes, examines emerging organelle-based therapies, and discusses key challenges related to safety, durability, and translation. Full article

(This article belongs to the Special Issue Pathogen–Host Interactions: Death, Defense, and Disease)

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36 pages, 3640 KB

Open AccessArticle

Bioinformatic Identification of CRISPR–Cas Systems in Leptospira Genus: An Update on Their Distribution Across 77 Species

by Ronald Guillermo Peláez Sánchez, Juanita González Restrepo, Santiago Pineda, Alexandra Milena Cuartas-López, Juliana María Martínez Garro, Marco Torres-Castro, Rodrigo Urrego, Luis Ernesto López-Rojas, Jorge Emilio Salazar Florez and Fernando P. Monroy

Pathogens 2025, 14(10), 1044; https://doi.org/10.3390/pathogens14101044 - 16 Oct 2025

Viewed by 530

Abstract

Leptospirosis is a globally distributed zoonotic disease caused by pathogenic bacteria of the Leptospira genus. Genome editing in Leptospira has been difficult to perform. Currently, the functionality of the CRISPR-Cas system has been demonstrated in species such as Leptospira interrogans. However, the [...] Read more.

Leptospirosis is a globally distributed zoonotic disease caused by pathogenic bacteria of the Leptospira genus. Genome editing in Leptospira has been difficult to perform. Currently, the functionality of the CRISPR-Cas system has been demonstrated in species such as Leptospira interrogans. However, the different CRISPR-Cas systems present in most of the 77 species are unknown. Therefore, the objective of this study was to identify these arrays across the genomes of all described Leptospira species using bioinformatics tools. Methods: a bioinformatics workflow was followed: genomes were downloaded from the NCBI database; Cas protein detection was carried out using the CRISPR-CasFinder and RAST web servers; functional analyses of Cas proteins were performed with InterProScan, ProtParam, Swiss Model, Alphafold3, Swiss PDB Viewer, and Pymol; conservation pattern detection was conducted using MEGA12, and Seqlogos; spacer identification was carried out with the Actinobacteriophages database and BLAST version 1.4.0; and bacteriophage detection was performed using PHASTER, and PHASTEST. Results: Cas proteins were detected in 36 out of the 77 species of the Leptospira species, including Cas1 to Cas9 and Cas12. These proteins were classified into Class 1 and Class 2 systems, corresponding to types I, II, and V. Direct repeats and spacers were detected in 19 species, with the direct repeats exhibiting two conserved nucleotide motifs. Analysis of spacer sequences revealed 323 distinct bacteriophages. Additionally, three intact bacteriophages were detected in the genomes of four Leptospira species. Notably, two saprophytic species have complete CRISPR-Cas systems. Conclusions: The presence of Cas proteins, direct repeats, and spacer sequences with homology to bacteriophage genomes provides evidence for a functional CRISPR-Cas system in at least 19 species. Full article

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17 pages, 462 KB

Open AccessReview

Human Papillomavirus: An Old New History

by Nicole West, Valentina Boz, Nunzia Zanotta, Carolina Cason, Giuseppina Campisciano, Alessandra Casuccio, Daniele Gianfrilli, Teresa Maria Assunta Fasciana, Giuseppina Capra, Maria Cristina Salfa, Franz Sesti, Barbara Suligoi, Francesca Valent, Laura Brunelli and Manola Comar

Pathogens 2025, 14(10), 1043; https://doi.org/10.3390/pathogens14101043 - 14 Oct 2025

Viewed by 772

Abstract

Human papillomavirus (HPV) represents the most common sexually transmitted infection worldwide and a major public health challenge. Nearly all sexually active individuals will acquire HPV during their lifetime, with the highest prevalence observed in adolescents and young adults shortly after sexual debut. More [...] Read more.

Human papillomavirus (HPV) represents the most common sexually transmitted infection worldwide and a major public health challenge. Nearly all sexually active individuals will acquire HPV during their lifetime, with the highest prevalence observed in adolescents and young adults shortly after sexual debut. More than 200 genotypes have been described, ranging from low-risk types, mainly responsible for benign lesions, to high-risk types, which are associated with cervical, anogenital, and head and neck cancers. While most infections are transient and spontaneously cleared by the immune system, persistent high-risk HPV can lead to precancerous lesions and malignant transformation, often in synergy with other sexually transmitted pathogens or in the context of microbiome imbalance. The introduction of vaccines and advanced screening technologies has substantially modified prevention strategies. Vaccination coverage remains heterogeneous, with persistent gaps particularly among males due to cultural, social, and educational barriers. Schools are increasingly recognized as strategic environments to promote awareness, sex education, and gender-neutral vaccination. Innovative approaches such as microbiome modulation, therapeutic vaccines, and liquid biopsy biomarkers are emerging as promising perspectives. This review aims to provide an updated overview of HPV epidemiology, clinical impact, prevention strategies, and future frontiers, with special attention to adolescents as a priority target group. Full article

(This article belongs to the Special Issue Focus on Cervicovaginal Health: Microbiome, Sexually Transmitted Infections, and Beneficial Microbes: 2nd Edition)

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19 pages, 6927 KB

Open AccessArticle

Serum Proteomics Reveals Systemic Responses in Didelphis aurita Naturally Infected with Hepatozoon sp.

by Andrés Mauricio Ortega Orozco, Camilo Jose Ramirez-Lopez, Lucas Drumond Bento, Pollyanna Cordeiro Souto, Fabrícia Modolo Girardi, Veronica Rodrigues Castro, Edvaldo Barros, Joao Vitor Gonçalves de Oliveira, Renner Philipe Rodrigues Carvalho, Artur Kanadani Campos and Leandro Abreu da Fonseca

Pathogens 2025, 14(10), 1042; https://doi.org/10.3390/pathogens14101042 - 14 Oct 2025

Viewed by 444

Abstract

Didelphis aurita is a widely distributed neotropical marsupial frequently found in peri-urban environments and known to harbor various pathogens, including hemoparasites of the genus Hepatozoon. However, the systemic physiological responses of naturally infected individuals remain poorly understood. This study aimed to characterize the [...] Read more.

Didelphis aurita is a widely distributed neotropical marsupial frequently found in peri-urban environments and known to harbor various pathogens, including hemoparasites of the genus Hepatozoon. However, the systemic physiological responses of naturally infected individuals remain poorly understood. This study aimed to characterize the serum proteomic profile of Didelphis aurita naturally infected with Hepatozoon sp., providing insights into host–parasite interactions and potential biomarkers of infection. Serum samples were analyzed using liquid chromatography–tandem mass spectrometry (LC-MS/MS), followed by functional annotation based on Gene Ontology and KEGG pathway enrichment. A total of 67 proteins were identified, 33 of which were exclusive to infected animals. The most abundant proteins included albumin, hemoglobin subunits, and venom metalloproteinase inhibitors (DM43 and DM64). Functional enrichment revealed significant involvement in complement and coagulation cascades, protease inhibition, antioxidant defense, and extracellular vesicle localization. Key proteins such as fibrinogen, plasminogen, antithrombin, SERPIN family members, vitronectin, and fibronectin suggest an integrated host response involving hemostasis, inflammation control, and tissue remodeling. This is the first report of the serum proteome of Didelphis aurita naturally infected with Hepatozoon sp. Despite the absence of protein validation, the findings provide novel insights into marsupial immunophysiology and offer a foundation for future biomarker research and ecoimmunological surveillance in synanthropic species. Full article

(This article belongs to the Special Issue Proteomic Approaches in Different Animal Pathogens)

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Pathogens, Volume 14, Issue 10 (October 2025) – 115 articles

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