Pathogens

15 pages, 1604 KB

Open AccessArticle

Host-Filtered Blood Nucleic Acids for Pathogen Detection: Shared Background, Sparse Signal, and Methodological Limits

by Zhaoxia Wang, Guangchan Chen, Mei Yang, Saihua Wang, Jiahui Fang, Ce Shi, Yuying Gu and Zhongping Ning

Pathogens 2026, 15(1), 55; https://doi.org/10.3390/pathogens15010055 - 6 Jan 2026

Abstract

Plasma cell-free RNA (cfRNA) metagenomics is increasingly explored for blood-based pathogen detection, but the structure of the shared background “blood microbiome”, the reproducibility of reported signals, and the practical limits of this approach remain unclear. We performed a critical re-analysis and benchmarking (“stress [...] Read more.

Plasma cell-free RNA (cfRNA) metagenomics is increasingly explored for blood-based pathogen detection, but the structure of the shared background “blood microbiome”, the reproducibility of reported signals, and the practical limits of this approach remain unclear. We performed a critical re-analysis and benchmarking (“stress test”) of host-filtered blood RNA sequencing data from two cohorts: a bacteriologically confirmed tuberculosis (TB) cohort (n = 51) previously used only to derive host cfRNA signatures, and a coronary artery disease (CAD) cohort (n = 16) previously reported to show a CAD-shifted “blood microbiome” enriched for periodontal taxa. Both datasets were processed with a unified pipeline combining stringent human read removal and taxonomic profiling using the latest versions of specialized tools Kraken2 and MetaPhlAn4. Across both cohorts, only a minority of non-host reads were classifiable; under strict host filtering, classified non-host reads comprised 7.3% (5.0–12.0%) in CAD and 21.8% (5.4–31.5%) in TB, still representing only a small fraction of total cfRNA. Classified non-host communities were dominated by recurrent, low-abundance taxa from skin, oral, and environmental lineages, forming a largely shared, low-complexity background in both TB and CAD. Background-derived bacterial signatures showed only modest separation between disease and control groups, with wide intra-group variability. Mycobacterium tuberculosis-assigned reads were detectable in many TB-positive samples but accounted for ≤0.001% of total cfRNA and occurred at similar orders of magnitude in a subset of TB-negative samples, precluding robust discrimination. Phylogeny-aware visualization confirmed that visually “enriched” taxa in TB-positive plasma arose mainly from background-associated clades rather than a distinct pathogen-specific cluster. Collectively, these findings provide a quantitative benchmark of the background-dominated regime and practical limits of plasma cfRNA metagenomics for pathogen detection, highlighting that practical performance is constrained more by a shared, low-complexity background and sparse pathogen-derived fragments than by large disease-specific shifts, underscoring the need for transparent host filtering, explicit background modeling, and integration with targeted or orthogonal assays. Full article

(This article belongs to the Section Bacterial Pathogens)

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8 pages, 538 KB

Open AccessArticle

Characterizing the Use of High-Dose Amoxicillin for the Treatment of Bacteremia

by Julia Lloyd, Kathleen Lau, Cindy San, Victor Leung and Colin Lee

Pathogens 2026, 15(1), 54; https://doi.org/10.3390/pathogens15010054 - 6 Jan 2026

Abstract

Treatment of bacteremia has traditionally consisted of a 7–14-day course of intravenous (IV) antibiotics. Transitioning from IV to oral (PO) antibiotics in uncomplicated cases of Gram-negative and Gram-positive bacteremia is non-inferior to a complete course of IV antibiotics. High-dose oral amoxicillin has been [...] Read more.

Treatment of bacteremia has traditionally consisted of a 7–14-day course of intravenous (IV) antibiotics. Transitioning from IV to oral (PO) antibiotics in uncomplicated cases of Gram-negative and Gram-positive bacteremia is non-inferior to a complete course of IV antibiotics. High-dose oral amoxicillin has been used in practice for treating bacteremia but has limited safety and efficacy data. We conducted a retrospective chart review between June 2022 and June 2024 to characterize the use of high-dose amoxicillin and evaluate its efficacy and safety. A convenient sample size of 100 patients was used. Patients admitted to hospital who received at least one dose of high-dose amoxicillin (1 g PO TID) for the treatment of bacteremia were included. Patients undergoing hemodialysis and patients receiving amoxicillin for other infections were excluded. The average patient was a 60-year-old male (66% male) with a Gram-positive respiratory or skin source bacteremia. The median time to transition to oral amoxicillin was 5 days. The median duration of total treatment was 14 days. Respiratory sources were treated for a shorter duration, whereas skin sources were treated for longer. Readmission to hospital occurred in 28% of cases. The majority of readmissions were unrelated to the original infection, and 92% of patients were cured. There were no observed adverse events, bacteremia relapses, or deaths. In this observational study, transitioning to high-dose oral amoxicillin was primarily used for treatment of uncomplicated respiratory and skin infections with secondary bacteremia. A high rate of clinical success was observed with high-dose PO amoxicillin, with no adverse events reported. Full article

(This article belongs to the Section Bacterial Pathogens)

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16 pages, 1084 KB

Open AccessArticle

Antigenic-Specificity and Cytokine Profile of the T-Cell Response to Human Cytomegalovirus in Transplant Recipients

by Federica Zavaglio, Paola Zelini, Asja Cera, Piera d’Angelo, Marilena Gregorini, Teresa Rampino, Lucia Del Frate, Federica Meloni, Oscar Borsani, Carlo Pellegrini, Fausto Baldanti and Daniele Lilleri

Pathogens 2026, 15(1), 53; https://doi.org/10.3390/pathogens15010053 - 5 Jan 2026

Abstract

Human cytomegalovirus (HCMV) infection is a significant complication in transplant recipients. Following HCMV reactivation, the recovery of T-cell responses serves as a key indicator of protection from HCMV disease. This study aimed to assess the HCMV-specific CD4⁺ and CD8⁺ T-cell responses [...] Read more.

Human cytomegalovirus (HCMV) infection is a significant complication in transplant recipients. Following HCMV reactivation, the recovery of T-cell responses serves as a key indicator of protection from HCMV disease. This study aimed to assess the HCMV-specific CD4⁺ and CD8⁺ T-cell responses and their cytokine production (IFNγ, TNFα, IL2) against various HCMV proteins (IE-1, pp65, gB, gH/gL/pUL128L) in solid organ transplant recipients (SOTRs) and hematopoietic stem cell transplant recipients (HSCTRs) with active HCMV infection. The cohort consisted of 16 SOTR and 16 HSCTR categorized into two groups: (i) Controllers, who spontaneously controlled the infection, and (ii) Non-Controllers, who required antiviral treatment. T-cell responses were analyzed following stimulation with peptide pools and intracellular cytokine staining. Prior to transplantation, all patients exhibited a significantly higher frequency of CD4⁺ T cells specific to pp65 compared to gH and gL/pUL128L. During the peak of infection, T-cell frequencies across all peptides were similar, but at infection resolution, the frequency of pp65 and gB-specific CD4⁺IFNγ⁺ T cells was significantly higher than gL/pUL128L. Additionally, pp65 and IE-1-specific CD8⁺IFNγ⁺ T-cell responses were significantly greater than those against gH and gL/pUL128L at the resolution of infection. Notably, Controllers exhibited significantly higher frequencies of monofunctional pp65-specific T cells, particularly in CD8⁺ T cells producing IFNγ and TNFα. The response to pp65, especially IFNγ production, may serve as a key marker for identifying patients capable of controlling HCMV infection. Full article

(This article belongs to the Special Issue Human Cytomegalovirus: From Molecular Pathogenesis to Therapeutic Innovations)

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13 pages, 3268 KB

Open AccessArticle

Potential Novel Genotype of “Bopivirus B” from Sheep in Türkiye: Epidemiology and Molecular Characterization

by Feray Alkan, İlke Karayel-Hacıoğlu, Selda Duran-Yelken, Fruzsina Tóth, Buket Pekşen and Ákos Boros

Pathogens 2026, 15(1), 52; https://doi.org/10.3390/pathogens15010052 - 5 Jan 2026

Abstract

Various microbial agents have been found in the feces of both humans and animals, especially in newborns. While some of these agents are recognized as causing diarrhea, the role of others, specifically bopiviruses of the family Picornaviridae, in diarrhea remains uncertain. In [...] Read more.

Various microbial agents have been found in the feces of both humans and animals, especially in newborns. While some of these agents are recognized as causing diarrhea, the role of others, specifically bopiviruses of the family Picornaviridae, in diarrhea remains uncertain. In this study, we conducted an analysis of 214 fecal samples from cattle (n = 114), sheep (n = 82), and goats (n = 18) with diarrhea, collected from farms across 17 different provinces in Türkiye. All samples were tested using RT-PCR targeting the 3D^(RdRp) region of bopiviruses, and two samples from sheep (2.4%) tested positive. The 7303 nt-long complete coding sequence of Bopivirus/Sheep/KS-1M/2024/TUR and partial 3D^(RdRp), VP3, and 2A-2C sequences of Bopivirus/Sheep/ANK-K30/2017/TUR were determined by additional RT-PCR, 3′RACE-PCR reactions and Sanger sequencing. Both strains show close sequence and phylogenetic relationship to members of species “Bopivirus B” of genus Bopivirus. Bopivirus/Sheep/KS-1M/2024/TUR is most closely related to a sheep Bopivirus B strain (sheep/14-73/2018/ITA) from Italy, but the phylogenetic separation, the low sequence identities and high p-distance values in VP1 to existing genotypes of “B1” and “B2” suggest that both strains could belong to novel genotypes (“B3” and “B4”) in species “Bopivirus B”, although additional closely related sequences are necessary for proper typing. Full article

(This article belongs to the Special Issue New Insights into Viral Infections of Domestic Animals)

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28 pages, 3309 KB

Open AccessReview

Immune Dysregulation in HIV-TB Co-Infection: Role of Cytokines and T Cell Biomarkers—A Narrative Review

by Catherine Keiko Gunawan, Anton Sumarpo and Agnes Rengga Indrati

Pathogens 2026, 15(1), 51; https://doi.org/10.3390/pathogens15010051 - 3 Jan 2026

Abstract

Immune dysregulation is a hallmark of human immunodeficiency virus (HIV) infection, characterized by persistent immune activation and systemic inflammation that drive T cell exhaustion and senescence, contributing to disease progression and non-AIDS comorbidities, most notably tuberculosis (TB). With rising HIV prevalence, the incidence [...] Read more.

Immune dysregulation is a hallmark of human immunodeficiency virus (HIV) infection, characterized by persistent immune activation and systemic inflammation that drive T cell exhaustion and senescence, contributing to disease progression and non-AIDS comorbidities, most notably tuberculosis (TB). With rising HIV prevalence, the incidence of HIV-TB co-infection continues to rise, highlighting the need to understand their immunopathological interplay. This narrative review aims to examine the association between immune dysregulation in HIV-TB co-infection, with a focus on cytokine profiles and immunological biomarkers. Relevant literature was retrieved from multiple databases, with evidence demonstrating differential expression of cytokines—IL-17A, IFN-γ, TNF, IL-10, IL-6, IL-4, and IL-2—and T cell activation markers, such as CD38 and HLA-DR on CD4⁺ T cells in latent and active TB among HIV-infected individuals. These immune mediators are consistently co-expressed at higher levels in active TB compared to latent TB, suggesting heightened immune activation of both innate and adaptive immune responses in HIV-TB co-infection. However, these findings are largely based on observational data, and the precise mechanism by which cytokine and T cell biomarker dysregulation contributes to HIV-TB pathogenesis remains incompletely understood, underscoring the need for larger, mechanistic studies to address these gaps in the pathogenic pathway. Full article

(This article belongs to the Special Issue HIV/AIDS Co-Infections and Non-AIDS Co-Morbidities)

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Graphical abstract

12 pages, 2806 KB

Open AccessSystematic Review

A Meta-Analysis of Influencing Factors for Reinfection of Hand, Foot and Mouth Disease in China, Based on Adjusted Effect Estimates

by Anmin Ge, Weihong Cui, Siyu Qu, Ning Wang, Wenhua Zhang, Lili Wei, Shuqin Zhou, Quanman Hu, Liquan Zhang and Shuaiyin Chen

Pathogens 2026, 15(1), 50; https://doi.org/10.3390/pathogens15010050 - 2 Jan 2026

Abstract

Background: Numerous studies have reported on the epidemiology of hand, foot and mouth disease (HFMD) reinfection and its potential influencing factors; however, findings regarding reinfection rates as well as determinants such as gender, age, residence, and pathogens remain inconsistent. Due to this heterogeneity [...] Read more.

Background: Numerous studies have reported on the epidemiology of hand, foot and mouth disease (HFMD) reinfection and its potential influencing factors; however, findings regarding reinfection rates as well as determinants such as gender, age, residence, and pathogens remain inconsistent. Due to this heterogeneity in reported outcomes, a comprehensive systematic review and meta-analysis are warranted to consolidate existing evidence. Methods: Effect estimates were expressed as reinfection rates, odds ratio (OR)/hazard ratio (HR) and 95% confidence intervals (CI). When necessary, data were converted to ensure consistency across comparison groups. Results: A thorough search was carried out using the predetermined literature retrieval approach across the PubMed, Web of Science, and Embase databases. Finally, 9 articles met the inclusion criteria and were included in this study. The results indicated that the overall reinfection rate for HFMD was 4.1% (95% CI: 2.0–6.2%). Males compared to females (overall effect = 1.256, 95% CI: 1.176–1.341), younger compared to older children (overall effect = 2.972, 95% CI: 1.512–5.843), scattered children compared to students (overall effect: 4.017, 95% CI: 1.560–10.344), and enterovirus 71 (EV71) compared to non-EV71 enteroviruses (overall effect = 0.71, 95% CI: 0.59–0.86) were associated with the HFMD reinfection. Conclusions: The overall HFMD reinfection rate was 4.1% (95% CI: 2.0–6.2%). Male, younger age, kindergarten children, and infection with non-EV71 enteroviruses (compared to EV71), were identified as significant risk factors for recurrent HFMD. Targeted intervention strategies should be developed for these high-risk populations to effectively reduce the incidence of reinfection. Full article

(This article belongs to the Section Epidemiology of Infectious Diseases)

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14 pages, 967 KB

Open AccessArticle

Evaluating the Performance of Loop-Mediated Isothermal Amplification for the Detection of Listeria monocytogenes Biofilms on Stainless Steel Surfaces

by Carmen Pilar Garrido-Pérez, Marta López-Cabo and Alejandro Garrido-Maestu

Pathogens 2026, 15(1), 49; https://doi.org/10.3390/pathogens15010049 - 1 Jan 2026

Abstract

L. monocytogenes is the causative agent of human listeriosis, a deadly disease with fatality rates up to 20%. L. monocytogenes has the ability to grow under harsh environmental conditions. It can form biofilms in food industries, making it capable of persisting in facilities. [...] Read more.

L. monocytogenes is the causative agent of human listeriosis, a deadly disease with fatality rates up to 20%. L. monocytogenes has the ability to grow under harsh environmental conditions. It can form biofilms in food industries, making it capable of persisting in facilities. Given this scenario, it is of utmost importance to rapidly detect this bacterium not only in foods but also on food-contact surfaces. For the successful outcome of any given detection technology, it is imperative to properly process the samples. In the present work, PBS, LPT, and LPT-Pronase were compared to determine which one could provide better results in DNA-based detection. Additionally, the effect of a short TSB pre-enrichment was assessed. To better mimic a real scenario, L. monocytogenes monospecies and multispecies biofilms were analyzed. It was observed that supplementing LPT with pronase, a protein-degrading enzyme, could better detach the biofilm, which achieved a 0.5 cycle reduction compared to the other broths, and the pre-enrichment reduced the real-time PCR by ~2 cycles. The samples were analyzed by real-time PCR and colorimetric LAMP, and the same results were obtained with both techniques regardless of the concentration of L. monocytogenes present in the biofilm; the initial concentration was 1.8 log CFU/cm² 15 min after the pre-enrichment. The results were confirmed by real-time PCR, which demonstrated the applicability of the methodology to be applied in decentralized setups, such as food-processing facilities, with minimal laboratory infrastructure. Full article

(This article belongs to the Special Issue Diagnosis, Immunopathogenesis and Control of Bacterial Infections)

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12 pages, 861 KB

Open AccessArticle

Enterovirus-D68—Neglected Pathogen in Acute Respiratory Infections: Insights from Croatia

by Zeljka Hruskar, Lucija Skara Abramovic, Ivana Ferencak, Dragan Juric, Josipa Lozic, Anita Juric, Bojana Bocka, Marin Bajek, Mirela Josipovic, Viktor Bekic and Irena Tabain

Pathogens 2026, 15(1), 48; https://doi.org/10.3390/pathogens15010048 - 1 Jan 2026

Abstract

Background: Enterovirus-D68 (EV-D68) was long underreported, with only sporadic cases of respiratory disease worldwide until 2014, when numerous countries experienced significant outbreaks of EV-D68. In Croatia, sporadic detections have primarily resulted from an absence of systematic surveillance. Following the increased incidence of EV-D68 [...] Read more.

Background: Enterovirus-D68 (EV-D68) was long underreported, with only sporadic cases of respiratory disease worldwide until 2014, when numerous countries experienced significant outbreaks of EV-D68. In Croatia, sporadic detections have primarily resulted from an absence of systematic surveillance. Following the increased incidence of EV-D68 across Europe in 2022, we started to characterize EV-positive respiratory samples in Zagreb to confirm the presence of EV-D68 and identify circulating lineages through phylogenetic analysis. Methods: Respiratory samples from individuals with acute respiratory infection and additional clinical symptoms were tested at the Virology Laboratory of the Croatian Institute of Public Health, and EV-positive samples were further screened using the real-time RT-qPCR method for EV-D68. VP1 sequences were obtained by sequencing and subsequently genotyped. Results: Between March 2022 and December 2024, EV was detected in 2048 respiratory samples. Annual distributions of EV detections were 656 (10.0%) in 2022, 785 (8.1%) in 2023, and 607 (7.4%) in 2024. EV-D68 was identified in 13.1% of EV-positive samples in 2022, 1.4% in 2023, and 19.6% in 2024. The peaks in EV-D68 circulation were observed in July (n = 24) and September (n = 24) in 2022 and in September 2024 (n = 62). Phylogenetic analysis of EV-D68 VP1 sequences revealed the presence of two major clades, A2 and B3. The sequences from 2022 clustered exclusively within clade B3, while in 2024 A2 clade was newly introduced. Conclusions: We confirmed the presence of EV-D68 in Croatia with circulating lineages corresponding to those detected elsewhere in Europe. The absence of routine testing has likely led to an underestimation of EV-D68 prevalence. These findings underscore the urgent need for ongoing surveillance and genomic characterization to clarify EV-D68 epidemiology in Croatia. Full article

(This article belongs to the Special Issue Emerging and Neglected Pathogens in the Balkans)

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26 pages, 2345 KB

Open AccessReview

Oral Route Infection by Trypanosoma cruzi: From the Beginning to the Present Day

by Sebastián Zambrano, Kurt Montoya, Alejandro Avalos, Bessy Gutiérrez, Juan San Francisco, José Luis Vega and Jorge González

Pathogens 2026, 15(1), 47; https://doi.org/10.3390/pathogens15010047 - 1 Jan 2026

Abstract

Trypanosoma cruzi is the causative agent of Chagas disease, which affects 6–7 million people worldwide. Although the possibility of oral transmission was first scientifically suggested in 1913, it was not until 1968 that the first confirmed cases of human infection via food consumption [...] Read more.

Trypanosoma cruzi is the causative agent of Chagas disease, which affects 6–7 million people worldwide. Although the possibility of oral transmission was first scientifically suggested in 1913, it was not until 1968 that the first confirmed cases of human infection via food consumption were reported. This long gap contributed to the widespread perception that oral transmission was a rare or incidental event. Over the past two decades, significant advances have been made in understanding the biological and clinical aspects of oral transmission, including the molecular mechanisms by which metacyclic trypomastigotes establish infection via the digestive route. Experimental studies in murine models have further deepened our knowledge of the biology and pathogenesis of oral infection. Concurrently, multiple outbreaks of T. cruzi infection through contaminated food and beverages have been reported across Latin America, providing valuable insights into the molecular epidemiology and clinical characteristics of this transmission route. Moreover, experimental evidence has shown that the consumption of meat from animals infected during the acute phase can also lead to T. cruzi infection, highlighting carnivory as a potential alternative transmission mechanism. This review aims to comprehensively analyze oral infection by T. cruzi, considering clinical and epidemiological data, parasite biology, and findings from murine experimental models. Strategies for controlling foodborne transmission of Chagas disease are also discussed. Full article

(This article belongs to the Special Issue Virulence and Molecular Cell Biology of Parasites)

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15 pages, 2163 KB

Open AccessArticle

Metabolomic Insights into MYMV Resistance: Biochemical Complexity in Mung Bean Cultivars

by Sudha Manickam, Veera Ranjani Rajagopalan, Madhumitha Balasubramaniam, Karthikeyan Adhimoolam, Senthil Natesan and Raveendran Muthurajan

Pathogens 2026, 15(1), 46; https://doi.org/10.3390/pathogens15010046 - 31 Dec 2025

Abstract

Yellow Mosaic Disease (YMD) caused by mungbean yellow mosaic virus (MYMV, begomovirus) is one of the main causes of low mungbean (Vigna radiata L.) productivity, primarily in South Asia. Agroinoculation screening for MYMV resistance in mungbean cultivar VGGRU 1, an interspecific derivative [...] Read more.

Yellow Mosaic Disease (YMD) caused by mungbean yellow mosaic virus (MYMV, begomovirus) is one of the main causes of low mungbean (Vigna radiata L.) productivity, primarily in South Asia. Agroinoculation screening for MYMV resistance in mungbean cultivar VGGRU 1, an interspecific derivative of mungbean × rice bean and VRM (Gg)1 across replications, revealed VGGRU1 as highly resistant to MYMV infection. Gas chromatography mass spectrometry analysis was performed on the methanolic leaf extracts of susceptible and resistant genotypes, along with necessary controls. The metabolite profiling of the susceptible and resistant genotypes, along with controls, identified 121 discriminant metabolites belonging to 24 different classes of metabolites. A maximum number of 27 metabolites were accumulated in agroinoculated VGGRU1 alone. Metabolite profiles of VGGRU1 and VRM1 were clustered hierarchically and revealed substantial variations between the genotypes. Fold change revealed the upregulation of amino acids and phenol in the resistant genotype. The resistant genotype, VGGRU1, showed significantly higher levels of key defense-related metabolites, such as amino acids and phenolics. In this study, 18 significant VIP metabolites were identified, differentiating the resistant VGGRU1 and susceptible VRM (Gg)1 genotypes. Full article

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

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22 pages, 510 KB

Open AccessReview

Diagnostic Accuracy of Multiplex NAAT/PCR and Culture Against Salmonella spp.: A Comparison of Meta-Analytical Methods

by Xanthoula Rousou, Luis Furuya-Kanamori, Eleftherios Meletis, Olympia Lioupi, Nikolaos Solomakos, Polychronis Kostoulas and Suhail A. R. Doi

Pathogens 2026, 15(1), 45; https://doi.org/10.3390/pathogens15010045 - 31 Dec 2025

Abstract

Background: Non-typhoidal (NT) Salmonella spp. constitutes a major cause of foodborne illness. Culture is the gold standard, but it is time consuming, whereas multiplex nucleic acid amplification tests (NAATs)/Polymerase Chain Reaction (PCR) offer faster detection with variable reported performance. Objectives: To compare the [...] Read more.

Background: Non-typhoidal (NT) Salmonella spp. constitutes a major cause of foodborne illness. Culture is the gold standard, but it is time consuming, whereas multiplex nucleic acid amplification tests (NAATs)/Polymerase Chain Reaction (PCR) offer faster detection with variable reported performance. Objectives: To compare the diagnostic accuracy of multiplex NAAT/PCR and culture for Salmonella spp. using various statistical models with or without a gold standard assumption. Methods: A systematic search (PubMed, Web of Science, Scopus; up to April 2024) identified 44 studies (55 comparisons). Diagnostic performance was evaluated using the frequentists bivariate model (BM) and Split Component Synthesis (SCS) and the Bayesian bivariate models (BBMs) and hierarchical summary ROC (BHSROC). Results: Across models, multiplex NAAT/PCR demonstrated high specificity (>98%) but model-dependent variability in sensitivity (85.5–94.8%), consistently substantial between study heterogeneity and threshold variation. The BM and BBM yielded a higher sensitivity estimate with narrower non-overlapping confidence intervals while SCS and BHSROC models, which are more robust to threshold differences, produced more conservative estimates with wider uncertainty. In Bayesian latent class analyses, culture remained highly accurate (Se: 97.17%, 95% CrI: 70.3–99.99; Sp: 96.06%, 95% CrI: 78.9–99.99), but with wide credible intervals indicating variation between studies, perhaps due to the different protocols used. Conclusion: Model choice affects inferred diagnostic accuracy, particularly when high heterogeneity is present. Both multiplex NAAT/PCR and culture showed high accuracy; hence, a combination of the two tests could optimise rapid diagnosis and treatment. Future research should include cost effectiveness and decision analysis to update the diagnostic algorithms. Full article

(This article belongs to the Special Issue Diagnosis, Immunopathogenesis and Control of Bacterial Infections)

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13 pages, 1948 KB

Open AccessArticle

Genetic Diversity of Infectious Bronchitis Virus Genotype II in Poland

by Anna Pikuła, Anna Lisowska, Justyna Opolska and Katarzyna Domańska-Blicharz

Pathogens 2026, 15(1), 44; https://doi.org/10.3390/pathogens15010044 - 31 Dec 2025

Abstract

The epidemiological situation in Poland for IBV GII (formerly known as D1466) has seemed stable over the years, but an increase in such infections has been recently reported. In this study, genetic characterization of the representatives of this genotype was performed in order [...] Read more.

The epidemiological situation in Poland for IBV GII (formerly known as D1466) has seemed stable over the years, but an increase in such infections has been recently reported. In this study, genetic characterization of the representatives of this genotype was performed in order to determine whether the new epidemic wave of GII IBV was responsible for changes in this status quo. Genotyping based on the complete S1 coding region of eight Polish IBV field strains from 2011 to 2021 confirmed that they belonged to genotype II, with two of them clustered in the two previously identified GII-1 and GII-2 lineages. In turn, the S1 coding region sequences of the next six Polish strains are very different from the previous ones and form a separate group on the phylogenetic tree. However, comprehensive analysis of all complete S1 coding regions of GII strains did not fulfill all parameters needed to create the separate GII lineage, and they all seem to belong to the GII-1 lineage. Further analysis of the partial S1 sequence of 15 IBV GII strains showed their genetic distinctiveness and indicates the ongoing evolution of this virus genotype. Considering the results of our study and the recent outbreaks of GII-2 in Western Europe, it appears that infections with GII virus strains mainly affect egg-producing, long-lived chickens, commercial layers, and breeders. Furthermore, due to the high diversity of these viruses, their circulation in the poultry population may remain undetected, and for this reason, the observed production problems in laying flocks may be attributed to other, unrelated factors. Full article

(This article belongs to the Special Issue Molecular Detection and Characterisation of Viral Pathogens: 2nd Edition)

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15 pages, 1280 KB

Open AccessArticle

Oral Microbiota Alterations and Potential Salivary Biomarkers in Colorectal Cancer: A Next-Generation Sequencing Study

by Salih Maçin, Özben Özden, Rugıyya Samadzade, Esra Saylam, Nurullah Çiftçi, Uğur Arslan and Serdar Yormaz

Pathogens 2026, 15(1), 43; https://doi.org/10.3390/pathogens15010043 - 30 Dec 2025

Abstract

Colorectal cancer (CRC) has a high mortality rate worldwide. Oral and intestinal microbiota members may have an effect on gastrointestinal tumors’ pathogenesis, particularly in CRC. Designed as a pilot study, this study’s aim was to investigate the relationship between CRC and oral microbiota [...] Read more.

Colorectal cancer (CRC) has a high mortality rate worldwide. Oral and intestinal microbiota members may have an effect on gastrointestinal tumors’ pathogenesis, particularly in CRC. Designed as a pilot study, this study’s aim was to investigate the relationship between CRC and oral microbiota and to identify potential biomarkers for CRC diagnosis. Saliva samples were collected from recently diagnosed CRC patients (n = 14) and healthy controls (n = 14) between March 2023 and December 2023. Microbiota (16S rRNA) analyses were conducted on these saliva samples using a next-generation sequencing method. Phylogenetic analyses, including alpha diversity, principal component analysis (PCA), principal coordinate analysis (PCoA), beta diversity, biomarker, and phenotype analyses, were conducted using the Qiime2 (Quantitative Insights Into Microbial Ecology) platform. Alpha diversity indices (Shannon: p = 0.78, Cho1: p = 0.28, Simpson: p = 0.81) showed no significant difference between CRC and control groups. Beta diversity analysis using Bray–Curtis PCoA indicated significant differences in the microbial community between the two groups (p = 0.003). Examination of OTU distributions revealed that the Mycoplasmatota phylum was undetectable in the oral microbiota of healthy controls but was significantly elevated in CRC patients (CRC: 0.13 ± 0.30, Control: 0.00 ± 0.00, p < 0.05). Additionally, Metamycoplasma salivarium, Bacteroides intestinalis, and Pseudoprevotella muciniphila were undetectable in healthy controls but significantly more prevalent in CRC patients (p < 0.05 for all three species). LEfSe analysis identified eight species with an LDA score > 2, Granulicatella adiacens, Streptococcus thermophilus, Streptococcus gwangjuense, Capnocytophaga sp. FDAARGOS_737, Capnocytophaga gingivalis, Granulicatella elegans, Bacteroides intestinalis, and Pseudoprevotella muciniphila, as potential biomarkers. The results of this study contribute critical evidence of the role of oral microbiota in the pathogenesis of colorectal cancer. Alterations in the microbiota suggest potential biomarkers in understanding the biological mechanisms underlying CRC and developing diagnostic and therapeutic strategies. Full article

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14 pages, 282 KB

Open AccessReview

Current Evidence on Safety, Efficacy and Efficiency of Sublingual Vaccine Uromune^® in Prevention of Recurrent Urinary Tract Infections: A Literature Review

by José Emilio Hernández-Sánchez, María Fernanda Lorenzo-Gómez and Carmen González-Enguita

Pathogens 2026, 15(1), 42; https://doi.org/10.3390/pathogens15010042 - 30 Dec 2025

Abstract

Recurrent urinary tract infections (rUTIs) are a prevalent public health problem in women, with significant clinical, psychological, and economic consequences. Standard antibiotic prophylaxis, while effective, is limited in the medium term due to the risk of bacterial resistance and potential side effects. This [...] Read more.

Recurrent urinary tract infections (rUTIs) are a prevalent public health problem in women, with significant clinical, psychological, and economic consequences. Standard antibiotic prophylaxis, while effective, is limited in the medium term due to the risk of bacterial resistance and potential side effects. This narrative review summarizes the current evidence on the efficacy, safety, and economic impact of Uromune^®—a sublingual bacterial vaccine—as a preventive strategy for rUTIs. A literature search was conducted, focusing on systematic reviews and meta-analyses that evaluated Uromune^® in women and in other specific at-risk populations. Available data show that Uromune^® reduces the frequency of UTI episodes, prolongs recurrence-free intervals, and decreases overall antibiotic use. The vaccine has a favorable safety profile, with predominantly mild adverse effects and a low discontinuation rate. Furthermore, its use has been associated with improved quality of life and a marked reduction in direct and indirect healthcare costs. These findings support Uromune^® as an effective, safe, and potentially cost-effective alternative to conventional antibiotic prophylaxis in the current context of increasing antimicrobial resistance. Full article

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

12 pages, 1042 KB

Open AccessArticle

High Occurrence of Pathogenic Free-Living Amoebae in Arid Environments

by Patricia Pérez-Pérez, Javier Chao-Pellicer, Rubén L. Rodríguez-Expósito, Marco Peña-Prunell, Angélica Domínguez-de-Barros, Omar García-Pérez, Elizabeth Córdoba-Lanús, María Reyes-Batlle, José E. Piñero and Jacob Lorenzo-Morales

Pathogens 2026, 15(1), 41; https://doi.org/10.3390/pathogens15010041 - 30 Dec 2025

Abstract

Free-living amoebae (FLA) are protozoa ubiquitous in nature, isolated from a variety of environments worldwide. In addition to their natural distribution, some species have been found to be pathogenic to humans. In the present study, FLA presence was evaluated and characterized at the [...] Read more.

Free-living amoebae (FLA) are protozoa ubiquitous in nature, isolated from a variety of environments worldwide. In addition to their natural distribution, some species have been found to be pathogenic to humans. In the present study, FLA presence was evaluated and characterized at the molecular level from different water and soil samples in Fuerteventura Island, Canary Islands, Spain. A total of 31 samples were analyzed by culture and molecular assays (q-PCR and PCR). Moreover, the microbiological quality of the water samples was examined as required by current legislation and international standards. The obtained data revealed that the genus Acanthamoeba was the most prevalent genus of FLA in soil samples and the species Vermamoeba vermiformis was the most isolated in water samples collected from Fuerteventura by culture and molecular assays, q-PCR, and conventional PCR/Sanger sequencing. On the other hand, a microbiological analysis revealed heterogeneous contamination patterns. Escherichia coli was detected in several samples, with some exhibiting high counts while others showed no presence. Salmonella spp. appeared in multiple samples, particularly FTVW1, FTVW9, and FTVW13, whereas Shigella spp. was only found in one sample (FTVW1). Moreover, q-PCR detection offers advantages such as reduced detection time and cost. In addition, culture was proven to be more effective for confirming FLA viability and isolating a greater variety of FLA. Overall, the occurrence of potentially pathogenic free-living amoebae in habitats related to the human population, as reported in the present study, supports the relevance of FLA as a potential health threat to humans. Full article

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

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17 pages, 2138 KB

Open AccessArticle

Five-Year Slaughterhouse-Based Surveillance of Echinococcus granulosus in Sheep from Yili, Northwest Xinjiang, China

by Cairen, Xiaoli Zhang, Li Zhang, Kalibixiati Aimulajiang, Batubayier Daoerji, Daoerji Namuka, Baoping Guo, Rongsheng Mi and Liying Wang

Pathogens 2026, 15(1), 40; https://doi.org/10.3390/pathogens15010040 - 29 Dec 2025

Abstract

Background: Cystic echinococcosis (CE) remains a significant zoonotic burden in the pastoral regions of China. Yili Prefecture in Xinjiang is a high-risk area, yet comprehensive data are lacking on the prevalence and molecular characteristics of Echinococcus granulosus in its primary intermediate host, sheep. [...] Read more.

Background: Cystic echinococcosis (CE) remains a significant zoonotic burden in the pastoral regions of China. Yili Prefecture in Xinjiang is a high-risk area, yet comprehensive data are lacking on the prevalence and molecular characteristics of Echinococcus granulosus in its primary intermediate host, sheep. Methods: From 2020 to 2024, a total of 2700 sheep livers were visually inspected for hydatid cysts infection at one randomly selected slaughterhouse in each of the nine counties of Yili Prefecture. Ninety cyst-positive samples were subjected to morphological examination and molecular genotyping by amplifying and sequencing the nad2 gene. Results: The overall prevalence of E. granulosus was 22.0% (594/2700). County-level prevalence ranged from 18.3% (Zhaosu County) to 25.7% (Huocheng County), with no significant differences observed among the counties (p > 0.05) except between Huocheng and Zhaosu. Temporally, the annual prevalence fluctuated between 20.2% and 24.2% without a consistent downward trend. Genotyping revealed that the G1 genotype was overwhelmingly dominant (95.2%, 79/83), with a minor circulation of the G3 genotype (4.8%, 4/83). Fourteen haplotypes were identified; Hap1 was the central and predominant haplotype (47.0%, 39/83), found in all counties. Network analysis suggested a recent population expansion of the parasite. Conclusion: This five-year surveillance study reveals a persistently high prevalence and complex genetic diversity of E. granulosus in sheep in Yili Prefecture. The dominance of the zoonotic G1 genotype indicates a substantial public health threat. Our findings provide crucial data for contributing to the development of local control strategies. However, the specific reasons for the high infection rate in sheep remain unclear, as this study did not include examinations of definitive hosts or environmental samples; this gap should be addressed in future research. Full article

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

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12 pages, 1549 KB

Open AccessArticle

Whole Genome Sequencing of Drug-Resistant Vibrio cholerae Serotype Ogawa from an Outbreak in Khyber Pakhtunkhwa

by Aftab Ali, Momin Khan, Taj Ali Khan, Sajjad Ahmad, Noor Rahman, Aiman Waheed and Taane G. Clark

Pathogens 2026, 15(1), 39; https://doi.org/10.3390/pathogens15010039 - 29 Dec 2025

Abstract

Background: Cholera, caused by Vibrio cholerae, remains endemic in many developing countries, including Pakistan. The extensive use of antibiotics has led to the emergence of antimicrobial resistance in V. cholerae, limiting available treatment options. In this study, we performed molecular characterisation [...] Read more.

Background: Cholera, caused by Vibrio cholerae, remains endemic in many developing countries, including Pakistan. The extensive use of antibiotics has led to the emergence of antimicrobial resistance in V. cholerae, limiting available treatment options. In this study, we performed molecular characterisation of antibiotic-resistant V. cholerae serotype Ogawa isolates from a recent cholera outbreak in Khyber Pakhtunkhwa, Pakistan. Methodology: Suspected cholera stool samples were collected from hospitalised patients at various district hospitals of Khyber Pakhtunkhwa Province (KPK), Pakistan. The samples were transported to the Public Health Reference Microbiology Laboratory at Khyber Medical University, Peshawar. V. cholerae were identified based on colonial morphology, Gram staining, and biochemical tests using EPI 10E. For serotype identification, monovalent antisera were used. Antibiotic susceptibility testing (AST) was performed using CLSI M45 and EUCAST guidelines. DNA was extracted from pure colonies of multidrug-resistant (MDR) V. cholerae and subjected to whole-genome sequencing (WGS) for genomic characterisation using an Illumina MiSeq platform. Results: Of the 350 active diarrheal cases investigated, 70 were confirmed as V. cholerae. The outbreak was initially reported in Dir and was subsequently followed by a high incidence of cholera in the Peshawar district of KPK. All strains belong to the Ogawa serotype, which shows high antibiotic resistance, particularly to ampicillin (n = 62, 88.57%), Sulfamethoxazole/Trimethoprim (n = 60, 85.71%), Erythromycin (n = 59, 84.29%), and Tetracycline (n = 53, 75.71%). The lowest resistance was against Meropenem (n = 1, 1.4%), followed by amikacin (n = 7, 10.0%) and levofloxacin (n = 13, 18.57%). Furthermore, 34 (48.57%) of the isolates were MDR, while 13 (18.57%) were extensively drug-resistant. Six samples were selected for whole-genome sequencing. The selection of six V. cholerae samples for WGS was based on their drug resistance pattern and origin of isolation. At the genomic level, all sequenced V. cholerae strains harboured multiple antimicrobial resistance determinants. Quinolone resistance was associated with mutations and genes in gyrA, gyrB, parC, and parE; resistance to sulfamethoxazole–trimethoprim with folA, folP, and dfr; tetracycline resistance with tetA and tet35; chloramphenicol resistance with catB and S10p; and aminoglycoside resistance with hns, S12p, and gigB. In addition, β-lactam resistance was linked to the presence of efflux and β-lactamase genes, including blaSHV and mox-3. Mutations were identified in gyrA at positions S83I, S177A, and S202A, and in parC at positions S85L and I231V. Collectively, the presence of these resistance determinants likely enables V. cholerae to survive exposure to high concentrations of multiple antibiotics. Conclusions: Our V. cholerae isolates showed close genetic relatedness to previously sequenced strains from Pakistan (2010 and 2022), as well as to recently reported international strains from the USA, Australia, and China. These findings highlight both the long-term persistence of these lineages within Pakistan and their international dissemination, likely facilitated by globalisation. Full article

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

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24 pages, 6620 KB

Open AccessArticle

No Evidence of Direct Transmission of Emerging Bluetongue Virus Strains Between Israel and Europe Based on Genomic Analyses (2013–2023)

by Natalia Golender, Eyal Klement and Bernd Hoffmann

Pathogens 2026, 15(1), 38; https://doi.org/10.3390/pathogens15010038 - 28 Dec 2025

Abstract

Bluetongue (BT) is an arthropod-borne viral disease primarily affecting domestic and wild ruminants. In recent years, several BTV serotypes and genotypes have been detected in Israel almost annually, raising questions about their origin and routes of introduction. Some BTV serotypes closely related to [...] Read more.

Bluetongue (BT) is an arthropod-borne viral disease primarily affecting domestic and wild ruminants. In recent years, several BTV serotypes and genotypes have been detected in Israel almost annually, raising questions about their origin and routes of introduction. Some BTV serotypes closely related to those first identified in Israel, including BTV-3, BTV-8, and BTV-12, were subsequently reported in Europe after a delay of several years. In this study, we sequenced the complete genomes of one representative strain of all newly identified Israeli BTV genotypes/serotypes—BTV-1, -4, -5, -8, and -11—first detected between 2021 and 2023. Additionally, complete sequences of enzootic Israeli BTV (2015) and eleven BTV-3 strains (2019–2023), with two representative strains for every year of isolation, except 2021 (three strains), were analyzed using phylogenetic, BLAST, and pairwise identity approaches. Genetic analyses revealed that recently identified Israeli and European BTV strains share common African ancestors, with some genomic “incursions” from Mayotte Island or the Arabian Peninsula. These incursions appeared more frequently in Israeli than in European strains. Nevertheless, nucleotide sequence differences of at least 2–3% across all genes indicate several years of independent evolution. The observed divergence suggests that no direct transmission of BTV occurred between Israel and Europe during the past decade. Full article

(This article belongs to the Special Issue Bluetongue and Other Orbiviruses)

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14 pages, 638 KB

Open AccessArticle

Time-Dependent Outcomes of Convalescent Plasma in Early COVID-19: A Single-Center Cohort with a Host–Pathogen Perspective

by Katarzyna Kalinowska, Patrycja Bociąga and Benita Wiatrak

Pathogens 2026, 15(1), 37; https://doi.org/10.3390/pathogens15010037 - 28 Dec 2025

Abstract

Background: Evidence on COVID-19 convalescent plasma (CCP) is mixed. We examined associations between CCP administration and in-hospital outcomes among patients hospitalized during early pandemic waves in Poland. Methods: We conducted a retrospective, single-center cohort study of adults hospitalized with COVID-19 between October 2020 [...] Read more.

Background: Evidence on COVID-19 convalescent plasma (CCP) is mixed. We examined associations between CCP administration and in-hospital outcomes among patients hospitalized during early pandemic waves in Poland. Methods: We conducted a retrospective, single-center cohort study of adults hospitalized with COVID-19 between October 2020 and January 2021. Patients receiving CCP were compared with contemporaneous controls without CCP. Primary outcomes were in-hospital mortality and discharge alive. Requirement for invasive mechanical ventilation/intubation was summarized descriptively because timing of intubation was not reliably available. Group comparisons used χ²/Fisher’s exact tests and t-test/Mann–Whitney U tests as appropriate. Associations with mortality and discharge were evaluated using logistic regression: (i) a prespecified age-adjusted model and (ii) an exploratory prognostic model including in-hospital treatments and severity markers (systemic glucocorticoids, remdesivir, oxygen therapy, and antibiotic use), interpreted prognostically rather than causally. Results: The cohort included 224 patients (CCP, n = 92; controls, n = 132); outcome status was missing for eight controls. Baseline demographics, comorbidities, and admission laboratory values were broadly comparable between groups. Crude in-hospital mortality was 25% in the CCP group (23/92) versus 42% in controls (52/124; p = 0.010), and discharge alive occurred in 66% versus 50%, respectively (p = 0.022). Invasive mechanical ventilation/intubation was required in 12.0% of CCP recipients and 4.5% of controls (p = 0.071). In age-adjusted models, CCP was associated with lower odds of in-hospital death. In exploratory prognostic models incorporating systemic glucocorticoids, remdesivir, oxygen therapy, and antibiotic use, CCP remained associated with lower odds of death and higher odds of discharge alive. Conclusions: In this early-wave retrospective cohort, CCP administration was associated with lower in-hospital mortality and higher discharge rates. Exploratory analyses adjusted for concomitant in-hospital therapies and severity markers should be interpreted as prognostic associations rather than evidence of causal efficacy. Full article

(This article belongs to the Special Issue Host-Pathogen Interactions During Pathogenic Human Coronavirus Infection: 2nd Edition)

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