Search Results (362)

African Swine Fever (ASF) control is severely limited by a diagnostic gap, as laboratory-based quantitative polymerase chain reaction (qPCR) is highly sensitive but slow, whereas field-deployable immunochromatographic assays (ICAs) are rapid but unreliable. To address this limitation, this study evaluated a novel, rapid isothermal assay, RNase hybridization-assisted amplification (RHAM), as a high-sensitivity, point-of-need diagnostic solution. This study compared the performance of RHAM and a conventional p72-based ICA against the qPCR reference standard using 106 diverse clinical field samples, including oral swabs, blood, serum, and organs, collected from suspected ASF cases in Thailand. The ICA exhibited markedly low diagnostic performance, achieving only 56.76% sensitivity and showing moderate agreement (κ = 0.421) with qPCR, highlighting the need for a more reliable alternative. In contrast, the RHAM assay achieved 94.59% sensitivity and 96.88% specificity, providing results rapidly within 35 min. This statistically superior performance (McNemar’s test, p < 0.0001) demonstrated almost perfect agreement (κ = 0.891) with the qPCR reference standard, missing only four samples with very high Ct values (>30). In conclusion, RHAM is a powerful, accurate, and field-deployable diagnostic tool that effectively bridges the diagnostic gap, offering qPCR-like sensitivity for the rapid containment of ASF outbreaks. Full article

Rural tropical regions face escalating threats from zoonotic AIV and dengue virus but lack sewered infrastructure for conventional wastewater surveillance. We implemented surface water-based surveillance (SWBS) in peri-urban Dhaka (Bangladesh) and Ruili (China) from July to November 2023 and coupled it with machine learning-enhanced digital epidemiology. Reverse transcription quantitative PCR (RT-qPCR) was employed to detect the M gene of AIV and to subtype H1, H5, H7, H9, and H10 in surface water. Wild bird feces (n = 40) were collected within 3 km of positive sites to source-track AIV. For the dengue virus, a serogroup-specific RT-qPCR assay targeting the CprM gene was used. Genomic sequencing of AIV and dengue virus was performed to elucidate phylogenetic relationships with local clinical strains. Clinical data related to dengue fever were also collected for correlation analysis. Meanwhile, 13 dengue-related keyword search volumes were harvested daily from Google, Bing and Baidu for four cities to reveal the relationship between dengue epidemics and the web search index. AIV H5 was detected in Dhaka city from week 38, peaking at week 39, while dengue virus was persistently detected from week 29 to week 45, aligning with clinical trends. Time-series cross-correlation analysis revealed that variations in surface water viral load led clinical case reports by approximately two weeks (max CCF = 0.572 at lag −2). In Ruili city, dengue virus was detected from week 32 to week 44. To sharpen sensitivity, 383 weekly web search series for 13 dengue keywords from four countries were screened; random-forest and XGBoost models retained five symptom queries that generated a composite index explaining 79% of variance in dengue RNA levels in an independent Ruili test set (n = 24) and reduced superfluous sampling by 35%. Phylogenetic analysis verified identity between water-derived and patient-derived DENV-2, confirming local transmission. The study demonstrates that AIV SWBS is optimally integrated with wild bird sampling for source attribution, whereas dengue SWBS achieves maximal efficiency when combined with real-time web search monitoring, providing tailored, low-cost early-warning modules for resource-constrained tropical settings. Full article

African swine fever (ASF), caused by African swine fever virus (ASFV), has inflicted severe economic losses on China’s pig industry. Existing ASFV nucleic acid detection methods struggle to identify infected pigs in the pre-viremic stage, especially for recently emerged recombinant ASFV strains that exhibit delayed clinical symptoms and prolonged virus shedding, posing great challenges to ASF prevention and control. To fit the problem, this study established a TaqMan duplex quantitative polymerase chain reaction (qPCR) assay targeting the ASFV p72 gene and porcine Hp gene for early diagnosis of ASFV infection. The qPCR reaction system (20 μL) and conditions were optimized and showed high sensitivity, with detection limits of 1.42 × 10¹ copies/μL for Hp and 2.23 × 10¹ copies/μL for ASFV, as well as excellent specificity and reproducibility. Serum cDNA samples from pigs infected with virulent or recombinant ASFV strains were tested, and the result showed that Hp was detectable as early as 1 day post-infection (DPI), however ASFV remained undetectable until 3DPI. Then cDNA samples from cohabitation infection were tested and 80% samples were Hp-positive, although ASFV test was negative.In conclusion, this duplex qPCR assay for simultaneous detection of Hp and ASFV enables pre-viremia diagnosis of ASF, providing a valuable tool for early screening of ASFV-infected pigs. Full article

Coxiella burnetii, the causative agent of human Q fever, subverts macrophage antimicrobial functions to establish an intracellular replicative niche. To better understand host–pathogen interactions, we investigated the transcriptional responses of human alveolar macrophages (hAMs) infected with virulent [NMI, G (Q212)], attenuated (NMII), and avirulent (Dugway) strains of C. burnetii. RNA sequencing indicated that all strains activated proinflammatory pathways, particularly IL-17 signaling, though the magnitude and nature of the response varied by strain. Infections with NMI, NMII or G (Q212) resulted in differential expression of roughly the same number of genes, while Dugway infection induced a stronger transcriptional response. Dugway and G (Q212) tended to polarize macrophages toward M1-like states, whereas responses to NMI and NMII were variable. Cytokine assays of NMII-infected THP-1 macrophages suggested the activation of IL-17 signaling, but only at later stages of infection, and single-cell RNA sequencing of NMII-infected THP-1 macrophages indicated heterogeneity in host response to infection, with distinct subpopulations exhibiting M1-like and M2-like inflammatory profiles. These findings highlight the complexity of macrophage response to C. burnetii and underscore the importance of strain-specific and cell-specific factors in shaping host immunity. Understanding these dynamics may inform the development of targeted therapies for Q fever. Full article

The Orthobunyavirus Oropouche (OROV) has become an urgent public health threat in Central and South America, as well as in other countries worldwide. Since its initial identification, there have been over 30 outbreaks, with the largest reported in late 2024 in Brazil. This outbreak prompted an epidemiological alert due to a significant increase in OF cases in non-Amazonian states in the Americas region, as well as in European countries, where 44 imported cases were identified. Humans become infected predominantly through the bite of the Culicoides paraensis midge, and the symptoms could be misinterpreted due to their similarity to those of other arboviral infections. Due to the lack of a point-of-care test, RT-qPCR is currently the key diagnostic test during the acute phase of the disease. This review focuses primarily on the available molecular and serological diagnostic methods. The latter could indeed be used as a confirmation test to monitor the patient’s immunological status and better distinguish between cross-reacting arboviruses. In addition, this review explains also the existing sequencing methods required to enforce the surveillance system for OROV reassortant species that could cause a new worldwide outbreak. The information gathered could provide a valuable basis for implementing additional surveillance systems in those countries lacking up-to-date data. Full article

Crimean-Congo hemorrhagic fever virus (CCHFV) is a tick-borne zoonotic pathogen of growing public health concern in southeastern Europe. This study provides the first serological evidence of CCHFV circulation in Croatia, based on testing 1473 serum samples from farm and companion animals, including sheep, horses, cattle, goats, dogs, and cats. A total of 109 samples (7.4%) tested positive for CCHFV antibodies using a commercially available enzyme-linked immunosorbent assay (ELISA) kit. The highest seroprevalence was recorded in sheep (28.3%), followed by horses (4.3%) and a single cat (0.5%), with no antibodies detected in cattle, goats, or dogs. Almost all seropositive animals originated from coastal and subcoastal Croatia, where Hyalomma ticks are present. Only two seropositive cases were detected in continental areas. Sheep samples from several farms in Zadar County showed intra-farm seropositivity rates of up to 85.7%, suggesting localised virus circulation likely influenced by vector distribution and farm-level practices. No viral ribonucleic acid (RNA) was detected by quantitative reverse transcription polymerase chain reaction (qRT-PCR), consistent with the transient nature of viremia in most animal hosts. These findings confirm the silent circulation of CCHFV in Croatia and reinforce the need for targeted, regionally adapted surveillance strategies that integrate multiple hosts and support early warning systems aligned with the One Health concept. Full article

Rift Valley fever (RVF) is a re-emerging vector-borne zoonotic disease that causes outbreaks in humans and animals across Africa. To better understand RVF at human–animal interfaces, a prospective longitudinal survey of people, livestock, and mosquitoes was conducted from 2016 to 2018, in two regions of Tanzania, with distinct climatic zones (Iringa and Morogoro). Molecular and serological tools for testing (RT-qPCR and IgM/IgG ELISA) for RVF virus (RVFV) were used to assess infection and exposure in people and animals. Mosquitoes were collected quarterly from 10 sentinel locations. In total, 1385 acutely febrile humans, 4449 livestock, and 3463 mosquito pools were tested. In humans, IgM seroprevalence was 3.75% (n = 52/1385), and overall seroprevalence (IgM and/or IgG positive) was 8.30% (n = 115/1385). People from Iringa had a higher exposure risk than those from Morogoro (aOR 2.63), and livestock owners had an increased risk compared to non-owners (aOR 2.51). In livestock, IgM seroprevalence was 1.09%, while overall seroprevalence was 10.11%. A total of 68.4% of herds had at least one seropositive animal. Sentinel animal follow-up revealed that the probability of seroconversion was significantly higher in Morogoro. Low-level RVFV RNA was detected in 8 human and 22 mosquito pools. These findings indicate active transmission among vectors, livestock, and people during the study period, highlighting the need for One Health surveillance approaches for RVFV and other arboviruses. Full article

Yellow fever (YF) is an acute and potentially fatal hemorrhagic disease caused by the Yellow Fever virus (YFV), endemic to sub-Saharan Africa and several tropical countries, including Brazil. In Brazil, the Amazon region is considered the main endemic area. YFV is maintained in a sylvatic cycle involving Neotropical primates and mosquitoes of the genera Haemagogus and Sabethes, acting as primary and secondary vectors, respectively. In March 2024, entomovirological surveillance was conducted in Santa Bárbara do Pará, Pará, Brazil. A total of 286 mosquitoes were collected, classified into 13 species across nine genera, and grouped into 33 pools. Seventeen pools were tested by RT-qPCR for Orthoflavivirus (YFV, DENV, WNV, SLEV), Alphavirus (CHIKV, MAYV), and Orthobunyavirus (OROV). YFV was detected in four Haemagogus janthinomys pools, with Ct values ranging from 22.2 to 27.9. Metagenomic sequencing confirmed the presence of YFV with assigned reads and >99% protein identity. Notably, the detection occurred without human cases or primate deaths, enabling timely vaccination of the local population. These findings confirm YFV circulation in forested areas of the Belém metropolitan region and reaffirm Hg. janthinomys as a key vector. Our study reinforces the relevance of early entomovirological surveillance and preventive strategies, such as vaccination, to mitigate yellow fever reemergence. Full article

Coxiella burnetii, the causative agent of Q fever, is a highly infectious pathogen capable of invading diverse cell types, from alveolar macrophages to trophoblasts. Within host cells, it establishes a replicative niche named Coxiella-containing vacuole (CCV). This is driven by effector proteins secreted by the bacterium into the host cell cytoplasm via a Type 4b Secretion System (T4SS). Advances in axenic culture and mutagenesis allowed the characterization of Coxiella effector proteins, revealing their host targets and strategies of cellular subversion. This review highlights recent insights into Coxiella effector proteins and their manipulation of host processes, from vesicular trafficking to innate immunity. Full article

Background: Q fever is a zoonotic disease caused by the intracellular bacterium Coxiella (C.) burnetii. In ruminants, it mainly leads to reproductive disorders. In humans, transmission typically occurs through direct contact with infected animals or inhalation of contaminated aerosols. Although it is a notifiable disease in the European Union for both humans and certain animal species, the actual incidence is likely underestimated due to the non-specific nature of clinical symptoms. Domestic ruminants are considered the main reservoirs of C. burnetii, placing farmers and veterinarians at increased occupational risk of infection. Objectives: This study aimed to assess the risk of Q fever infection in northern Italy by comparing the seroprevalence rates between professionally exposed individuals and not professionally exposed people. Methods: A total of 209 serum samples were analysed: 117 from exposed professionals (veterinarians, biologists, agronomists, laboratory technicians) and 92 from professionally unexposed people (control group). Serum samples were tested with a commercial enzyme-linked immunosorbent assay to detect the presence of IgG against C. burnetii. Positive and doubtful samples were further investigated with a commercial immunofluorescence assay for detection of IgM and IgG. Epidemiological data were also collected to explore potential risk factors. Results: In total, 10 of the 117 exposed individuals tested positive, yielding a seroprevalence of 8.6%, while only 1 of the 92 control subjects tested positive (1.1%). These findings indicate a significantly higher occupational risk of C. burnetii infection among exposed professionals compared to the general population. Conclusions: The results highlight the need for preventive measures and surveillance in at-risk occupational groups. Full article

Justicia procumbens L. (JP) has been traditionally used to treat colds with fever, swollen and sore throat, jaundice, malaria and eczema. Studies indicate that lignans constitute the primary bioactive components, yet systematic phytochemical investigations remain limited. Therefore, it is necessary to establish a rapid and effective method to identify the chemical components in JP. In this study, ultra-high-performance liquid chromatography-quadrupole-Exactive Orbitrap mass spectrometry (UHPLC-Q-Exactive Orbitrap MS) coupled with parallel reaction monitoring (PRM) was used for the first time to investigate JP. Based on chromatographic retention times, MS and MS² data, and bibliography data, a total of 132 compounds were tentatively identified, including 54 lignans, 19 flavonoids, 31 organic acids, 18 alkaloids, and 10 other types of constituents. Among these, 77 compounds are reported for the first time in JP, including 14 potential novel compounds. These results provide valuable reference and data support for the study of pharmacodynamic substances and quality control of this medicinal plant. Full article

Co-infection of Orientia tsutsugamushi and influenza A virus complicates diagnosis and treatment in endemic regions because of overlapping clinical features and potential synergistic inflammation. We describe a 68-year-old woman from Hainan, China, who presented with five days of high fever (39.2 °C), nonproductive cough, eschar formation, lymphadenopathy, cytopenias, elevated liver enzymes, and raised inflammatory markers. On the day of admission, influenza A was confirmed by rapid antigen test and Orientia tsutsugamushi IgM/IgG was detected via colloidal-gold immunochromatography, prompting concurrent oseltamivir and doxycycline therapy. Quantitative PCR on day 2 measured an Orientia tsutsugamushi load of 2.85 × 10⁴ copies/mL (Cq 28.86), and targeted next-generation sequencing on day 3 revealed a high H1N1pdm09 viral burden (>1 × 10⁶ copies/mL) with low-level human herpesvirus 1 co-detection. Nested PCR and Sanger sequencing assigned Orientia tsutsugamushi to the Karp_A lineage and influenza A to clade 6B.1A.5a.2a. The patient defervesced by hospital day 2, laboratory indices normalized by day 3, and radiographic abnormalities resolved by day 6. This first documented Orientia tsutsugamushi–influenza A co-infection in China highlights the value of integrating rapid serology, qPCR quantification, nested PCR genotyping, and tNGS for early, precise dual-pathogen identification. Systematic multi-pathogen screening during overlapping transmission seasons is recommended to guide timely combination therapy and enhance epidemiological surveillance. Full article

Background: Preschool-aged children can have difficulty adhering to infection control measures and were affected during the Omicron wave of the coronavirus disease 2019 (COVID-19) pandemic. However, the impacts of prior severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and vaccination on viral load in this age group remain poorly understood. This study aimed to investigate the relationship between previous SARS-CoV-2 infection, COVID-19 vaccination, and viral load or clinical severity in preschool-aged children infected during the Omicron variant epidemic in Japan. Methods: This prospective observational study investigated 107 children aged 1–75 months who were diagnosed with COVID-19 between May and September 2023. Rapid antigen (Ag) tests were performed on days 1 and 5 or 6, and results were visually graded into four categories (–, ±, 1+, or 2+). Ag results were validated against quantitative real-time reverse transcription polymerase chain reaction (RT-qPCR) cycle threshold (Ct) values. Clinical parameters, including vaccination status, previous infection, age, maximum body temperature, and fever duration, were analyzed using multivariate regression models. Results: Higher Ag loads (1+/2+) were more frequently observed in younger children who had not experienced prior infection or full vaccination. Prior infection and vaccination were independently linked to lower Ag loads and reduced maximum body temperature. Many unvaccinated and infection-naïve children continued to show elevated Ag levels on day 5 or 6, corresponding to Ct values suggestive of potential infectivity. Conclusions: Prior SARS-CoV-2 infection and vaccination were linked to lower viral loads and milder febrile responses among preschool-aged children. These findings enhance our understanding of infection dynamics in this age group and may inform future discussions on public health strategies in pediatric settings. Full article

Coxiella burnetii, the etiological agent of Q fever, is a globally distributed zoonotic pathogen affecting both animals and humans. Despite its known endemicity in various Mediterranean regions, data on human seroprevalence in Sardinia are still lacking. This study aimed to assess seroprevalence in patients and to analyze the annual positivity rate related to the serum samples collected in Sardinia over a ten-year period (2015–2024). For this purpose, a total of 1792 patients were involved in the survey, and 4310 serum samples were analyzed using indirect immunofluorescence assay (IFI) to detect IgM and IgG antibodies against C. burnetii. The global seroprevalence rates relating to all the patients over a ten-year period were determined along with the annual positivity rate and trends from all sera. An overall seroprevalence of 27.0% and an average of annual positivity rate of 16.0% were determined, with the IFI detecting IgG antibodies in 15.2% of positive samples and IgM antibodies in 0.9%, suggesting significant prior exposure of the population evaluated. Annual positivity rates ranged from 24.8% in 2016 to 8.0% in 2020. These results confirmed the endemic circulation of C. burnetii in Sardinia and the ongoing risk of human exposure. A GIS-based map was built to evidence the spatial distribution of Q fever in Sardinia. Interestingly, areas with higher seroprevalence appear to coincide with the distribution of sheep and goat farms, indicating a link between livestock and human exposure. These findings confirm the circulation of C. burnetii in Sardinia and underscore the importance of epidemiological monitoring, public health interventions, and educational efforts in populations at increased risk. Full article

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