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Crimean–Congo hemorrhagic fever (CCHF) is a zoonotic viral disease endemic to parts of Africa, Asia and southeastern Europe. Bulgaria is one of the few European countries with the consistent annual reporting of human CCHF cases. This study provides a descriptive overview of 24 confirmed CCHF cases in Bulgaria between 2015 and 2024. Laboratory confirmation was performed by an enzyme-linked immunosorbent assay (ELISA) and/or real-time reverse transcriptase polymerase chain reaction (RT-qPCR) testing. Common findings included fever, fatigue, gastrointestinal symptoms, thrombocytopenia, leukopenia, liver dysfunction and coagulopathy. Two fatal cases were recorded. Two samples collected in 2016 and 2024 were subjected to whole-genome sequencing. Phylogenetic analysis showed that both strains clustered within the Turkish branch of the Europe 1 genotype and shared high genetic similarity with previous Bulgarian strains, as well as strains from neighboring countries. These findings suggest the long-term persistence of a genetically stable viral lineage in the region. Continuous molecular and clinical surveillance is necessary to monitor the evolution and public health impact of CCHFV in endemic areas. Full article

To determine the association between FGFR4 (rs351855 and rs7708357) gene variants, serum levels, and immunohistochemical markers (Ki-67 and p53) in pituitary adenoma (PA), a case-control study was conducted involving 300 subjects divided into two groups: the control group (n = 200) and a group of PA (n = 100). The genotyping of FGFR4 rs351855 and rs7708357 was carried out using the real-time polymerase chain reaction (RT-PCR) method. The serum FGFR4 levels were measured using the ELISA method. Immunohistochemical analysis (Ki-67 and p53) was conducted. Statistical analysis of the data was performed using IBM SPSS Statistics 30.0 software. There were no statistically significant differences after analyzing the genotypes and alleles of FGFR4 rs351855 and rs7708357 in patients with PA and control groups (all p > 0.05). After evaluating the distribution of genotypes and alleles of FGFR4 rs351855 and rs7708357 in micro/macro, invasiveness, activity, and recurrence of PA and the control groups, the analysis showed no statistically significant differences between the groups (p > 0.05). Similarly, no significant differences in FGFR4 levels were observed between PA patients and control group (median (IQR): 3642.41 (1755.08) pg/mL vs. 3126.24 (1334.15) pg/mL, p = 0.121). Immunohistochemistry for Ki-67 revealed a labeling index (LI) of <1% in 25.5% of patients with PA, an LI of 1% in 10.9%, and an LI of >1% in 63.6% of patients. Further analyses showed no statistically significant associations with tumor size, invasiveness, activity, or recurrence. Immunohistochemistry for p53 revealed that macroadenomas had a significantly higher p53 H-score compared to microadenomas (median (IQR): 30.33 (28.68) vs. 18.34 (17.65), p = 0.005). Additionally, a moderate, statistically significant positive correlation between the Ki-67 LI and the p53 expression was found (Spearman’s ρ = 0.443, p = 0.003, n = 43). FGFR4 variants and serum protein levels were not significantly associated with PA risk or tumor features. Conversely, immunohistochemical markers Ki-67 and p53 were more informative, with higher p53 expression in macroadenomas and a moderate positive correlation between Ki-67 and p53, highlighting their potential relevance in tumor growth assessment. Full article

The problem of spreading harmful infections through contaminated surfaces has become more acute during the recent coronavirus pandemic. The design of self-cleaning materials, which can continuously decompose biological contaminants, is an urgent task for environmental protection and human health care. In this study, the surface of blended cotton/polyester fabric was functionalized with N-doped TiO₂ (TiO₂-N) nanoparticles using titanium(IV) isopropoxide as a binder to form durable photoactive coating and additionally decorated with Cu species to promote its self-cleaning properties. The photocatalytic ability of the material with photoactive coating was investigated in oxidation of acetone vapor, degradation of deoxyribonucleic acid (DNA) fragments of various lengths, and inactivation of PA136 bacteriophage virus and Candida albicans fungi under visible light and ultraviolet A (UVA) radiation. The kinetic aspects of inactivation and degradation processes were studied using the methods of infrared (IR) spectroscopy, polymerase chain reaction (PCR), double-layer plaque assay, and ten-fold dilution. The results of experiments showed that the textile fabric modified with TiO₂-N photocatalyst exhibited photoinduced self-cleaning properties and provided efficient degradation of all studied contaminants under exposure to both UVA and visible light. Additional modification of the material with Cu species substantially improved its self-cleaning properties, even in the absence of light. Full article

Background: Severe damage to one side of the brain often leads to adverse consequences and can also cause widespread changes throughout the brain, especially in the contralateral area. Studying molecular changes in the contralateral cerebral hemisphere, especially with regard to genetic regulation, can help discover potential treatment strategies to promote recovery after severe brain trauma on one side. Methods: In our study, the right motor cortex was surgically removed to simulate severe unilateral brain injury, and changes in glial cells and synaptic structure in the contralateral cortex were subsequently assessed through immunohistological, morphological, and Western blot analyses. We conducted transcriptomic studies to explore changes in gene expression levels associated with the inflammatory response. Results: Seven days after corticotomy, levels of reactive astrocytes and hypertrophic microglia increased significantly in the experimental group, while synapsin-1 and PSD-95 levels in the contralateral motor cortex increased. These molecular changes are associated with structural changes, including destruction of dendritic structures and the encapsulation of astrocytes by synapses. Genome-wide transcriptome analysis showed a significant increase in gene pathways involved in inflammatory responses, synaptic activity, and nerve fiber regeneration in the contralateral cortex after corticorectomy. Key transcription factors such as NF-κB1, Rela, STAT3 and Jun were identified as potential regulators of these contralateral changes. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) confirmed that the mRNA expression levels of Cacna1c, Tgfb1 and Slc2a1 genes related to STAT3, JUN, and NF-κB regulation significantly increased in the contralateral cortex of the experimental group. Conclusions: After unilateral brain damage occurs, changes in the contralateral cerebral hemisphere are closely related to processes involving inflammation and synaptic function. Full article

(1) The beach areas of Galveston, Texas, USA are heavily used for recreational activities and often experience elevated fecal indicator bacteria levels, representing a potential threat to ecosystem services, human health, and tourism-based economies that rely on suitable water quality. (2) During the span of 15 months (March 2022–May 2023), water samples that exceeded the U.S. Environmental Protection Agency-accepted alternative Beach Action Value (BAV) for enterococci of 104 MPN/100 mL were analyzed via microbial source tracking (MST) through quantitative polymerase chain reaction (qPCR) assays. The Bacteroides HF183 and DogBact as well as the Catellicoccus LeeSeaGull markers were used to detect human, dog, and gull fecal sources, respectively. The qPCR MST data were then utilized in a quantitative microbial risk assessment (QMRA) to assess human health risks. Additionally, samples collected in July and August 2022 were sequenced for 16S rRNA and matched with fecal sources through the Bayesian SourceTracker2 program. (3) Overall, 26% of the 110 samples with enterococci exceedances were positive for at least one of the MST markers. Gull was revealed to be the primary source of identified fecal contamination through qPCR and SourceTracker2. Human contamination was detected at very low levels (<1%), whereas dog contamination was found to co-occur with human contamination through qPCR. QMRA identified Campylobacter from canine sources as being the primary driver for human health risks for contact recreation for both adults and children. (4) These MST results coupled with QMRA provide important insight into water quality in Galveston that can inform future water quality and beach management decisions that prioritize public health risks. Full article

Despite intensive eradication efforts, classical swine fever (CSF) remains endemic across South America, Europe, Asia, and the Caribbean, highlighting the need for more effective surveillance and detection methods. Reverse-transcription real-time polymerase chain reaction (RRT-PCR) is the fastest, and most sensitive assay for detecting CSF virus (CSFV) genomic material. Previously, we demonstrated that spleen swabs outperformed spleen homogenates for the detection of ASFV genomic material by RRT-PCR. In this study, we compared CSFV genome detection in paired spleen homogenates and spleen swabs generated using 49 frozen and 33 fresh spleen samples collected from experimentally inoculated pigs with acute infection. The results show that the CSFV genome detection in spleen swabs is comparable to that in spleen homogenates. The study also demonstrated that the CSFV genomic material can be detected in spleen swabs during early CSFV infections, and the viruses can be successfully isolated from the swabs. The use of spleen swabs instead of spleen tissue homogenates for CSF detection will reduce labor, decrease costs associated with reporting, and increase the diagnostic throughput. Full article

Via a One Health approach, this study concomitantly assessed the susceptibility of humans and dogs to Trypanosoma cruzi infections on three islands and in two mainland seashore areas of southern Brazil. Human serum samples were tested using an enzyme-linked immunosorbent assay (ELISA) to detect anti-T. cruzi antibodies, while dog serum samples were tested using indirect fluorescent antibodies in an immunofluorescence assay (IFA). Seropositive human and dog individuals were also tested using quantitative polymerase chain reaction (qPCR) in corresponding blood samples. Overall, 2/304 (0.6%) human and 1/292 dog samples tested seropositive for T. cruzi by ELISA and IFA, respectively, and these cases were also molecularly positive for T. cruzi by qPCR. Although a relatively low positivity rate was observed herein, these cases were likely autochthonous, and the individuals may have been infected as a consequence of isolated events of disturbance in the natural peridomicile areas nearby. Such a disturbance could come in the form of a fire or deforestation event, which can cause stress and parasitemia in wild reservoirs and, consequently, lead to positive triatomines. In conclusion, T. cruzi monitoring should always be conducted in suspicious areas to ensure a Chagas disease-free status over time. Further studies should also consider entomological and wildlife surveillance to fully capture the transmission and spread of T. cruzi on islands and in seashore mainland areas of Brazil and other endemic countries. Full article

Glanders is a highly contagious and often fatal zoonotic disease of equids caused by Burkholderia mallei, a pathogen of significant concern due to its potential for bioterrorism. In Brazil, glanders remains endemic, particularly among working equids in the Northeast region. Diagnostic confirmation typically involves serology, culture, and polymerase chain reaction (PCR), although false-negative PCR results have been increasingly reported. This study aimed to evaluate the diagnostic performance and analytical sensitivity of four B. mallei-specific PCR primer sets using samples from 30 seropositive equids. Microbiological cultures were obtained from various organs and swabs, followed by PCR targeting four genomic regions: fliP-IS407A(a), fliP-IS407A(b), Burk457, and Bm17. All animals were confirmed positive for B. mallei via culture, but PCR detection rates varied significantly across primer sets. The fliP-IS407A(b) primer set showed the highest sensitivity, detecting 86% of samples, while the WOAH-recommended fliP-IS407A(a) set had the lowest performance (13.4%). Analytical sensitivity assays confirmed that fliP-IS407A(b) and Bm17 primers detected DNA concentrations as low as 0.007 ng, outperforming the others. These findings suggest that certain widely used primer sets may lack sufficient sensitivity for reliable detection of B. mallei, especially in chronically infected animals with low bacterial loads. The study underscores the need for ongoing validation of molecular diagnostics to improve the detection and control of glanders in endemic regions. Full article

Resistance to cytarabine (Ara-C) remains a major obstacle to the successful treatment of acute myeloid leukemia (AML). Therefore, modulating Ara-C resistance is indispensable for improving clinical outcomes. We previously demonstrated that sodium caseinate (SC), a salt derived from casein, the principal milk protein, inhibits proliferation and modulates the expression of Ara-C resistance-related genes in chemoresistant cells. However, it remains unclear whether the combination of SC with antineoplastic agents enhances apoptosis, modulates chemoresistance-related genes, and prolongs the survival of tumor-bearing mice implanted with chemoresistant cells. Here, we investigated the effects of SC in combination with Ara-C or daunorubicin (DNR) on cell proliferation, apoptosis, the expression of chemoresistance-associated genes, and the survival of tumor-bearing mice. Crystal violet assays, quantitative reverse transcription polymerase chain reaction (qRT-PCR), immunofluorescence, flow cytometry, and Kaplan–Meier survival curves were used to evaluate the effects of combinations in chemoresistant cells. We demonstrate that the IC₂₅ concentration of SC, when combined with antileukemic agents, increases the sensitivity of chemoresistant WEHI-CR50 cells to Ara-C by downregulating SIRT1 and MDR1, upregulating the expression of ENT1 and dCK, enhancing apoptosis, and prolonging the survival of WEHI-CR50 tumor-bearing mice. Our data suggest that SC in combination with antileukemic agents could be an effective adjuvant for Ara-C-resistant AML. Full article

Global warming and anthropogenic climate change are projected to expand the geographic distribution and population abundance of ectothermic species and exacerbate the biological invasion of exotic species. DNA methylation, as a reversible epigenetic modification, could provide a putative link between the phenotypic plasticity of invasive species and environmental temperature variations. We assessed and interpreted the epigenetic mechanisms of invasive and indigenous species’ differential tolerance to thermal stress through the invasive species Bemisia tabaci Mediterranean (MED) and the indigenous species Bemisia tabaci AsiaII3. We examine their thermal tolerance following exposure to heat and cold stress. We found that MED exhibits higher thermal resistance than AsiaII3 under heat stress. The fluorescence-labeled methylation-sensitive amplified polymorphism (F-MSAP) results proved that the increased thermal tolerance in MED is closely related to DNA methylation changes, other than genetic variation. Furthermore, the quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting analysis of DNA methyltransferases (Dnmts) suggested that increased expression of Dnmt3 regulates the higher thermal tolerance of female MED adults. A mechanism is revealed whereby DNA methylation enhances thermal tolerance in invasive species. Our results show that the Dnmt-mediated regulation mechanism is particularly significant for understanding invasive species’ successful invasion and rapid adaptation under global warming, providing new potential targets for controlling invasive species worldwide. Full article

Hepatitis E virus (HEV) is a leading cause of acute viral hepatitis and is primarily transmitted via contaminated water and food. Groundwater may also serve as a potential vector for HEV transmission. This study aimed to optimize real-time polymerase chain reaction (rtPCR) for the detection of HEV, employing both TaqMan probe- and SYBR Green-based methods. A total of 12 primer sets for the TaqMan probe-based method and 41 primer sets for the SYBR Green-based method were evaluated in order to identify the optimal combinations. Primer sets #4 (TaqMan probe-based) and #21 (SYBR Green-based) demonstrated the highest sensitivity and specificity, successfully detecting HEV in artificially spiked samples at 1 fg/μL. The TaqMan probe-based method facilitated rapid detection with minimized non-specific amplification, whereas the SYBR Green-based method allowed for broader primer exploration by leveraging melting curve analysis. Despite the absence of HEV detection in 123 groundwater samples, this study underscores the value of real-time PCR for environmental monitoring and paves the way for enhanced diagnostic tools for public health applications. Full article

Background: Polymerase chain reaction (PCR) is highly sensitive and specific for the rapid diagnosis of invasive fungal disease (IFD) but is not yet widely implemented due to concerns regarding limited standardisation between assays, the lack of commercial options and the absence of clear guidance on interpreting results. Objectives and Methods: This review provides an update on technical and clinical aspects of PCR for the diagnosis of the most pertinent fungal pathogens, including Aspergillus, Candida, Pneumocystis jirovecii, Mucorales spp., and endemic mycoses. Summary: Recent meta-analyses have demonstrated that quantitative PCR (qPCR) offers high sensitivity for diagnosing IFD, surpassing conventional microscopy, culture and most serological tests. The reported specificity of qPCR is likely underestimated due to comparison with imperfect reference standards with variable sensitivity. Although the very low limit of detection of qPCR can generate false positive results due to procedural contamination or patient colonisation (particularly in pulmonary specimens), the rates are comparable to those observed for biomarker testing. When interpreting qPCR results, it is essential to consider the pre-test probability, determined by the patient population, host factors, clinical presentation and risk factors. For patients with low to moderate pre-test probability, the use of sensitive molecular tests, often in conjunction with serological testing or biomarkers, can effectively exclude IFD when all tests return negative results, reducing the need for empirical antifungal therapy. Conversely, for patients with high pre-test probability and clinical features of IFD, qPCR testing on invasive specimens from the site of infection (such as tissue or bronchoalveolar lavage fluid) can confidently rule in the disease. The development of next-generation sequencing methods to detect fungal infection has the potential to enhance the diagnosis of IFD, but standardisation and optimisation are essential, with improved accessibility underpinning clinical utility. Full article

Background: Acute myeloid leukemia (AML) is a common and aggressive adults hematological malignancies. This study explored megakaryocyte–erythroid progenitors (MEPs) signature genes and constructed a prognostic model. Methods: Uniform manifold approximation and projection (UMAP) identified distinct cell types, with differential analysis between AML-MEP and normal MEP groups. Univariate and the least absolute shrinkage and selection operator (LASSO) Cox regression selected biomarkers to build a risk model and nomogram for 1-, 3-, and 5-year survival prediction. Results: Ten differentially expressed genes (DEGs) related to overall survival (OS), six (AHSP, MYB, VCL, PIM1, CDK6, as well as SNHG3) were retained post-LASSO. The model exhibited excellent efficiency (the area under the curve values: 0.788, 0.77, and 0.847). Pseudotime analysis of UMAP-defined subpopulations revealed that MYB and CDK6 exert stage-specific regulatory effects during MEP differentiation, with MYB involved in early commitment and CDK6 in terminal maturation. Finally, although VCL, PIM1, CDK6, and SNHG3 showed significant associations with AML survival and prognosis, they failed to exhibit pathological differential expression in quantitative real-time polymerase chain reaction (qRT-PCR) experimental validations. In contrast, the downregulation of AHSP and upregulation of MYB in AML samples were consistently validated by both qRT-PCR and Western blotting, showing the consistency between the transcriptional level changes and protein expression of these two genes (p < 0.05). Conclusions: In summary, the integration of single-cell/transcriptome analysis with targeted expression validation using clinical samples reveals that the combined AHSP-MYB signature effectively identifies high-risk MEP-AML patients, who may benefit from early intensive therapy or targeted interventions. Full article

Introduction: This study aimed to assess the accuracy of real-time polymerase chain reaction (PCR) as a diagnostic tool for Pneumocystis jirovecii pneumonia (PCP) in immunocompromised patients and evaluate the applicability of quantification cycle (Cq) data for PCP diagnosis. Methods: Clinical and laboratory data were collected from medical records of 96 immunocompromised patients hospitalized at the Hadassah hospital from 2018 to 2022, for lower respiratory tract infection. PCP diagnosis was independently categorized by two infectious disease specialists, blinded to PCR results, as either “definite” (confirmed by microscopic identification of P. jirovecii) or “probable” (compatible clinical data and negative microscopy). Clinical characteristics, PCR test performance, and Cq values were then compared between these PCP diagnostic groups and a control group of 85 patients who underwent bronchoscopy for indications unrelated to P. jirovecii infection. Results: The PCR test was found to be highly reliable for diagnosing PCP, with high sensitivity and specificity (93.1%, 98.7%, respectively), a positive predictive value (PPV) of 96.4%, a negative predictive value (NPV) of 97.1%, a negative likelihood ratio of 0.71, and a positive likelihood ratio of 46.5. A Cq cutoff value of 21.89 was found to discriminate between probable PCP and definite PCP. In addition, patients with probable PCP had lower in-hospital mortality than those with definite PCP or no PCP. Conclusions: PCR offers a promising approach for diagnosing PCP in immunocompromised patients with negative respiratory microscopy results. While further research may be warranted, its use may allow for more timely treatment and potentially improved outcomes. Full article

Background and Aims: The majority of individuals with COVID-19 developed acute symptoms. Post-COVID-19 syndrome refers to the signs and symptoms of COVID-19 that persist for more than 12 weeks. The present study was conducted to estimate the prevalence and risk factors for post-COVID-19 syndrome in the Omani population. Methods: This is a cross-sectional study that was conducted at the University Hospital Center (UHC). All patients diagnosed with COVID-19 (through polymerase chain reaction PCR testing) between March 2020 and March 2022 were included. Eligible participants were interviewed through a phone call, informed about the study procedure, and invited to participate in the study. Results: The study enrolled 265 COVID-19 patients, of whom 156 (59.2%) were females and 204 (77.3%) had been vaccinated. The overall prevalence of post-COVID-19 syndrome was 48.5%. The most common symptom was fatigue (71, 26.9%), followed by joint pain (44, 16.7%). The other symptoms included loss of taste/smell (34, 12.9%), cough (32, 12.1%), palpitation (25, 9.5%), and hair loss (27, 10.2%). Unvaccinated patients showed a higher incidence of fatigue (p = 0.03) and loss of smell/taste (p = 0.01) on univariate analysis. Females were at high risk for the development of various symptoms, including fatigue, muscular pain, breathing difficulty, cough, chest pain, palpitation, headache, and hair loss. Multivariate analysis showed that female gender is a significant independent predictor (odds ratio: 3.1; p = 0.00) for the development of post-COVID-19 syndrome. Conclusions: The prevalence of post-COVID-19 syndrome among the Omani population was high, highlighting the need for targeted interventions to manage long-term symptoms in vulnerable groups. Full article