Search Results (191)

Identifying pathogens causing periprosthetic joint infection (PJI) is a challenge for clinicians. We aimed to evaluate the application of metagenomic next-generation sequencing (mNGS) to identify pathogens in PJI. A prospective analysis was conducted of patients diagnosed PJI between 2022 and 2024 at twelve hospitals in Taiwan. Both conventional bacterial culture (CMT) and mNGS of joint fluid and debrided tissue were performed. Demographic characteristics, laboratory results and clinical outcomes were collected. The diagnostic performance of these two methods was analyzed. A total of 42 patients with a mean age of 67.9 years were enrolled in analysis. The knee was the most common joint involved (69.1%). A high proportion of patients (78.6%) received prior antibiotics within the two weeks at sample collection. mNGS identified pathogens in 28 out of 42 patients (66.7%), whereas CMT yielded positive results in 12 out of 42 patients (28.6%) (McNemar’s test, p = 0.01). Staphylococcus species was the most common genus detected (n = 11), followed by Cutibacterium (n = 4). Other detected genera included Escherichia, Mycobacterium, Enterobacter, Klebsiella (n = 2 each), Acinetobacter, and Corynebacterium (n = 1 each). Our results support the idea that mNGS could serve as a valuable diagnostic tool for PJI in addition to traditional culture methods. Full article

Astroviruses are non-enveloped, positive-sense single-stranded RNA viruses known to infect various mammals and birds, including humans, often causing gastrointestinal disorders. In recent years, astroviruses have also been linked to neurological and respiratory diseases across several species, including ruminants, mink, deer, and other mammals. Notably, astrovirus infections in goats have been documented in countries such as Switzerland and China, where novel genotypes have been identified in fecal samples. However, their role in the context of disease remains unclear, and reports focusing solely on goat astrovirus in the United States have not been published. A necropsy case of a Boer goat kid with a history of diarrhea was submitted for investigation following death in January 2025. Fresh tissues were received and used for histopathology and enteric pathogen testing, including parasitic, bacterial, and viral workups. Metagenomic-based next-generation sequencing (mNGS) was also applied for this case. Histological examination revealed severe necrotizing enterocolitis. The small intestine exhibited epithelial ulcerations, villus atrophy, hyperplastic and dilated crypts with necrotic debris, few intraenterocytic coccidian parasites, and increased inflammatory cells in the lamina propria. The large intestine showed similar findings with pleomorphic crypt enterocytes. Standard enteric pathogen tests were negative except for aerobic culture that identified Escherichia.coli and Enterococcus hirae. mNGS and bioinformatic analysis identified a novel astrovirus in the intestinal content that showed the highest nucleotide identity (86%) to the sheep strain Mamastrovirus 13 sheep/HA3 from China based on BLAST analysis. Phylogenetic analysis indicated that the newly identified caprine astrovirus IL90175 clustered with astrovirus strains from small ruminants in Asia and Europe. This research reports the discovery, histopathologic features, and genetic characteristics of a gastrointestinal disease-causing astrovirus in a goat kid, which had not been previously described in the United States. Full article

In 2020, a dairy farm in northwest Germany reported several cows with severe respiratory disease, fever, and reduced milk production. Multiple direct and indirect diagnostic methods were used to identify the cause of the disease. However, the pathogens detected could not be correlated with the severity of the clinical symptoms, so further diagnostic steps were taken. Blood and nasal swab samples were examined using next-generation sequencing (NGS) as part of a metagenomic analysis. For the first time in Germany, Hepacivirus bovis genotype 2 was detected. Real-time RT-PCR assays confirmed the presence of BovHepV genotypes 1 and 2 in the herd between 2020 and 2023. Analyses of complete and partial genome sequences demonstrated the presence of different virus variants in the herd over several years. In addition, the sequence data indicated that cattle can be reinfected with viruses belonging either to different BovHepV subtypes or to the same subtype. Although no direct link could be established between the detection of bovine hepaciviruses and the observed clinical symptoms, the PCR and sequence data obtained provide valuable insights into the epidemiology and pathogenesis of BovHepV infections. Full article

Probe-based capture represents a highly sensitive and cost-effective approach for overcoming host background and enriching viruses in metagenomic NGS (mNGS) libraries. Using clinical specimens collected globally from patients with fever or respiratory illness, we generated mNGS libraries by random priming and Nextera XT tagmentation, followed by target enrichment (teNGS) with Comprehensive Viral Research Panel (CVRP) probes. Capture pool sizes and total reads were optimized, and libraries were initially sequenced separately. Using only 3–4% of reads required for standard mNGS, teNGS achieved increased sensitivity, 100–10,000× increases in depth, and >50% genome coverage for pathogens with titers ≥ 1000 cp/mL. Application to >2000 clinical specimens from various matrices and to contrived samples containing viruses absent from the CVRP probe set enabled detection of diverse viral families and established a minimum 65% nucleotide identity for hybridization, respectively. To save time and resources, teNGS and mNGS libraries were then combined into one sequencing run: teamNGS. In addition to streamlining the workflow, teamNGS also improved genome recovery. Coupling methods maintain the sensitivity and coverage for viruses achieved by enrichment alone while also ensuring comprehensive recovery of non-viral microbes. teamNGS has the potential to improve patient management and lower the rates of unnecessary testing and antibiotic use. Full article

Bloodstream infections (BSIs) pose a significant global health challenge, particularly due to the increasing prevalence of antimicrobial resistance (AMR). Timely and accurate identification of pathogens and resistance determinants is critical for guiding appropriate therapy and improving patient outcomes. Traditional culture-based diagnostics are limited by prolonged turnaround times and reduced sensitivity, especially in culture-negative or polymicrobial infections. This review systematically examined current and emerging sequencing technologies for AMR detection in BSIs, including whole-genome sequencing (WGS), targeted next-generation sequencing (tNGS), metagenomic next-generation sequencing (mNGS), and long-read sequencing platforms (Oxford Nanopore, PacBio). We compared their clinical performance using key metrics such as diagnostic sensitivity, turnaround time, and cost, highlighting contexts in which each technology is most effective. For example, tNGS can achieve the rapid detection of known resistance genes within 8–24 h, while WGS provides comprehensive genome-wide resistance profiling over 24–48 h. mNGS offers broader detection, including rare or unexpected pathogens, although at higher cost and longer processing times. Our analysis identifies specific strengths and limitations of each approach, supporting the use of context-specific strategies, such as combining rapid targeted sequencing for common pathogens with broader metagenomic approaches for complex cases, to improve diagnostic yield and guide antimicrobial therapy. Quantitative comparisons indicate that sequencing technologies can complement conventional methods, particularly in cases where culture-based approaches fail. In conclusion, sequencing-based diagnostics offer measurable improvements in sensitivity and speed over traditional methods for AMR detection in BSIs. Future work should focus on optimizing workflows, integrating sequencing data into clinical decision-making, and validating approaches in prospective studies. Full article

Bovine respiratory disease (BRD) is a multifactorial syndrome and a leading cause of morbidity and economic loss in global cattle production. Next-generation sequencing (NGS) platforms, including Illumina and Oxford Nanopore Technologies (ONT), have enabled high-resolution profiling of the bovine respiratory microbiome and virome, revealing novel viral contributors such as bovine rhinitis A virus (BRAV) and influenza D virus (IDV). Transcriptomic approaches, including RNA sequencing (RNA-Seq) and microRNA (miRNA) profiling, provide insights into host immune responses and identify potential biomarkers for disease prediction. Traditional diagnostic methods—culture, ELISA, and immunohistochemistry—are increasingly complemented by PCR-based and metagenomic techniques, improving sensitivity and specificity. Despite technological progress, gaps remain in virome characterization, miRNA function, and the integration of multi-omics data. Standardized protocols and longitudinal studies are needed to validate microbial signatures and support field-deployable diagnostics. Advances in bioinformatics, particularly network-based integrative pipelines, are becoming essential for harmonizing multi-omics datasets and revealing complex host–pathogen interactions. The objective of this comprehensive review was to synthesize current understanding of the bovine transcriptomic response to BRD as well as the respiratory microbiome and virome, emphasizing how advanced sequencing technologies have transformed microbial profiling and molecular diagnostics in BRD. Full article

Diarrhea in young ruminants is a global issue and causes significant economic losses worldwide. In addition to common pathogens like rotavirus, coronavirus, and astrovirus, new viruses can be identified through unbiased next-generation sequencing (NGS) techniques. Here, we report the initial identification of a hunnivirus from a one-month-old goat with diarrhea using shotgun metagenomic NGS. A complete hunnivirus genome was recovered. Phylogenetic tree analysis revealed that this goat hunnivirus was more closely related to cattle hunnivirus than to small ruminant hunnivirus strains, suggesting a prior cross-species transmission event. The genome was used to design primers/probes for the conserved 3D^pol RdRP gene for real-time RT-PCR to screen banked ruminant fecal samples. Screening of 144 ruminant fecal samples showed that 9 of 38 goat, 22 of 96 cattle, and 0 of 8 sheep samples were positive for hunnivirus. Sequencing of the 3D^po region was performed on selected positive samples and revealed two lineages of hunnivirus circulating in North America. Our study highlights the importance of further investigation and monitoring of fecal samples using unbiased metagenomic tools to identify potential pathogens or co-infections in ruminants. Full article

Metagenomic next-generation sequencing (mNGS) is increasingly employed for the diagnosis of lower respiratory tract infections (LRTIs). However, the relative diagnostic performance of long-read versus short-read sequencing platforms remains incompletely defined. For this systematic review, a search was conducted in PubMed, Embase, Scopus, Web of Science, and Google Scholar to identify studies directly comparing long-read (e.g., Oxford Nanopore, PacBio) and short-read (e.g., Illumina, Ion Torrent, BGISEQ) metagenomic sequencing for the diagnosis of LRTI. Eligible studies reported diagnostic accuracy or comparative performance between platforms. Risk of bias was evaluated using the QUADAS-2 tool. Thirteen studies met inclusion criteria. Reported platforms included Illumina, Oxford Nanopore, PacBio, Ion Torrent, and BGISEQ-500. A total of 13 studies met inclusion criteria. Across studies reporting sensitivity, average sensitivity was similar for Illumina (71.8%) and Nanopore (71.9%). Specificity varied substantially, ranging from 42.9 to 95% for Illumina and 28.6 to 100% for Nanopore. Concordance between platforms ranged from 56 to 100%. Illumina consistently produced superior genome coverage (approaching 100% in most reports) and higher per-base accuracy, whereas Nanopore demonstrated faster turnaround times (<24 h), greater flexibility in pathogen detection, and superior sensitivity for Mycobacterium species. Risk of bias was frequently high or unclear, particularly in patient selection (6 studies), index test interpretation (5), and flow and timing (4), limiting the robustness of pooled estimates. Long-read and short-read mNGS platforms exhibit comparable strengths in the diagnosis of LRTIs. Illumina remains optimal for applications requiring maximal accuracy and genome coverage, whereas Nanopore offers rapid, versatile pathogen detection, particularly for difficult-to-detect organisms such as Mycobacterium. However, there are certain limitations of the review, including a lack of comparable outcomes reported in all studies; therefore, further research is warranted to address this. Full article

Background: Balamuthia mandrillaris is a free-living amoebic parasite that primarily causes rare opportunistic infections in immunocompromised hosts. Balamuthia amoebic encephalitis (BAE) is a rare yet severe parasitic infection affecting the central nervous system. It has an extremely low incidence in China but can have a mortality rate as high as 98%. The clinical manifestations of amebic infections are similar to those of bacterial and tuberculous meningitis, lacking specificity, which makes accurate diagnosis challenging in the clinical setting. Case Presentation: A 61-year-old immunocompetent woman experienced worsening headache and a moderate fever over the course of five days, initially treated as a common cold. On 25 February 2025, she exhibited behavioral abnormalities, dysphagia, and a high fever of 40.2 °C, which progressed to a coma. On 26 February, her cranial CT scan revealed multifocal hemorrhagic lesions in the right frontotemporoparietal lobes. The MRI revealed similar lesions with slight enhancement and herniation. She underwent an emergency decompressive craniectomy, yet her condition continued to deteriorate following the surgery. On 27 February, serum targeted next-generation sequencing (tNGS) detected B. mandrillaris. Additionally, metagenomic NGS (mNGS) of the cerebrospinal fluid (CSF) sample confirmed the presence on 28 February. Finally, B. mandrillaris was identified through a brain tissue biopsy on 3 March. However, due to the delayed diagnosis and lack of effective drugs, her condition rapidly deteriorated and became irreversible. Her family ultimately chose to withdraw treatment. Conclusions: This study highlights the application of NGS for early diagnosis of patients with severe CNS infection. Both tNGS and mNGS can be considered for the rapid detection of rare or novel pathogens and for facilitating diagnosis. Full article

Viral metagenomic next-generation sequencing (vmNGS) has transformed our capacity for the untargeted detection and characterisation of (re)emerging zoonotic viruses, surpassing the limitations of traditional targeted diagnostics. In this review, we critically evaluate the current landscape of vmNGS, highlighting its integration within the One Health paradigm and its application to the surveillance and discovery of (re)emerging viruses at the human–animal–environment interface. We provide a detailed overview of vmNGS workflows including sample selection, nucleic acid extraction, host depletion, virus enrichment, sequencing platforms, and bioinformatic pipelines, all tailored to maximise sensitivity and specificity for diverse sample types. Through selected case studies, including SARS-CoV-2, mpox, Zika virus, and a novel henipavirus, we illustrate the impact of vmNGS in outbreak detection, genomic surveillance, molecular epidemiology, and the development of diagnostics and vaccines. The review further examines the relative strengths and limitations of vmNGS in both passive and active surveillance, addressing barriers such as cost, infrastructure requirements, and the need for interdisciplinary collaboration. By integrating molecular, ecological, and public health perspectives, vmNGS stands as a central tool for early warning, comprehensive monitoring, and informed intervention against (re)emerging viral threats, underscoring its critical role in global pandemic preparedness and zoonotic disease control. Full article

Ticks are important vectors of pathogens affecting humans and animals, posing a serious threat to health. For the first time, we studied the metagenomic profile of the microbial composition of Hyalomma scupense and Hyalomma asiaticum ticks in Kazakhstan. A total of 94 adult H. asiaticum and H. scupense ticks collected from randomly selected cattle in Kazakhstan in 2023 were analyzed. 16S rRNA gene sequencing was performed using the Ion Torrent NGS platform. Taxonomic classification was carried out in the BV-BRC platform with the Kraken2 database. Metagenomic analysis revealed 26 bacterial genera, including both pathogenic and symbiotic taxa. In H. scupense, the dominant groups were Francisella (89.0%), Staphylococcus (76.0%) and Candidatus Midichloria (61.0%), while in H. asiaticum, they were Francisella (99.0% and 95.0%) and Helcococcus (65.0%). In male H. scupense, the proportion of Francisella reached 89%, whereas in females, it varied from 2% to 28%. In H. asiaticum, Helcococcus accounted for 65% in males compared to 11% in females. This is the first report on the metagenomic profile of the microbiota of H. scupense and H. asiaticum in Kazakhstan. The detection of pathogens indicates a risk of their transmission to humans and animals and highlights the need to develop new tick control strategies. Full article

Background: Immunocompromised children with malignancies or after hematopoietic cell transplantation (HCT) often deteriorate before conventional cultures identify a pathogen. Next-generation sequencing (NGS) promises faster, broader detection, yet its clinical impact in pediatric oncology remains unclear. This review aimed to assess the diagnostic performance and clinical utility of NGS in this population. Methods: We searched PubMed, Embase, and Scopus from January 2010 to April 2025 for studies evaluating NGS (metagenomic, targeted, or whole-genome sequencing) in pediatric oncology or HCT patients meeting predefined eligibility criteria. Duplicate screening, data extraction, and Joanna Briggs Institute risk-of-bias appraisal were performed. Heterogeneity precluded formal meta-analysis; findings were synthesized using narrative synthesis complemented by limited quantitative analyses. The protocol was not registered. Results: Twenty-four studies (≥2700 children; 2019–2025) met inclusion criteria. Metagenomic NGS (mNGS) was the most common approach, applied to blood/plasma (46%), bronchoalveolar fluid (BALF) (21%), and other fluids. In culture-negative sepsis or persistent febrile neutropenia, mNGS detected pathogens in 69–86% of episodes versus 18–56% for culture/polymerase chain reaction (PCR). Described in limited studies, early (<48 h) testing shortened fever by ~1.5 days and cut antimicrobial costs by 25–30%. Across studies, treatment was escalated, de-escalated, or discontinued in a median of 63% of mNGS-positive cases. Whole-genome sequencing (WGS) identified 18 silent transmission clusters and resolved a multidrug-resistant Acinetobacter baumannii outbreak within hours. Conclusions: NGS benefits pediatric hemato-oncology by accelerating pathogen-directed therapy, supporting antimicrobial stewardship, and enhancing outbreak surveillance. Despite cost and standardization barriers, evidence supports its use in selected high-risk patients. Full article

Prostate cancer (PCa) is the most common genitourinary malignancy in men, with a multifactorial etiology influenced by genetic, environmental, and microbial determinants. Although the prostate was traditionally considered sterile, advances in microbiome research have challenged this view, revealing potential links between microbial communities and PCa development, progression, and treatment response. This review synthesizes evidence on the gut, urinary, seminal fluid, and prostatic microbiomes, highlighting their potential contributions to PCa pathogenesis and therapeutic outcomes. Key studies utilizing next-generation sequencing (NGS), whole-genome sequencing (WGS), PCR, and metagenomic analyses have identified specific bacterial and fungal taxa associated with Pca; however, findings remain inconsistent across methodologies and cohorts. Microorganisms such as Propionibacterium acnes and Pseudomonas spp. may modulate inflammation, immune responses, and resistance to androgen-deprivation therapy. Further research is required to determine whether microbial signatures can serve as reliable biomarkers for early detection, prognosis, or novel therapeutic strategies in PCa management. Full article

Background/Objectives: Ureaplasma parvum (Up) is an opportunistic pathogen associated with urogenital tract infections, pregnancy complications, and reproductive system diseases. Advances in molecular diagnostics have expanded its pathogenic spectrum to include invasive conditions such as arthritis, meningitis, and pneumonia. However, the pathogenic significance of Up remains controversial. Methods: This study retrospectively analyzed nine adult cases of Up detected by metagenomic next-generation sequencing (mNGS) between 2023 and 2024. Results: Patients, aged 21 to 70 years, predominantly had underlying immunosuppressive conditions (66.7%). Infections involved the urinary system (44.4%), respiratory system (33.3%), and peritoneal cavity (22.2%). Symptomatic relief was achieved in five cases following treatment with tetracyclines, quinolones or tigecycline. Conclusions: These findings highlight Up as a potential causative agent of invasive infections, particularly in immunocompromised patients. Up has potential pathogenic significance, whether it is detected as a single pathogen or as a coexisting pathogen. Full article

Background and Clinical Significance: Granulomatosis with polyangiitis (GPA), an immune-mediated systemic small-vessel vasculitis affecting the upper/lower respiratory tracts and kidneys, frequently presents with non-specific nasal symptoms that lead to misdiagnosis. Case Presentation: We report a case of a 55-year-old female with GPA complicated by Bartter syndrome. She presented with one month of left nasal congestion, rhinorrhea, epistaxis, and headache. Initial diagnosis was acute sinusitis. Computed tomography (CT) revealed left maxillary and ethmoid sinus inflammation with bone destruction, while metagenomic next-generation sequencing (mNGS) suggested conventional bacterial infection. Postoperative pathology demonstrated chronic mucosal inflammation with lymphoid tissue hyperplasia. GPA was ultimately diagnosed based on PR3-ANCA seropositivity and chest CT findings of cavitary pulmonary nodules. Postoperatively, severe hypokalemia and hypomagnesemia secondary to Bartter syndrome emerged. Following electrolyte correction, induction therapy with glucocorticoids and cyclophosphamide was initiated. Conclusions: This case underscores that GPA’s head and neck manifestations are frequently misdiagnosed as infections or malignancies. Early diagnosis requires vigilance for GPA ‘red flags’, such as refractory nasal symptoms to conventional therapy (e.g., bloody rhinorrhea), characteristic CT findings (e.g., sinus opacification without ostiomeatal complex obstruction), and nasal endoscopy findings (e.g., ulcers/crusting). Otolaryngologists play a pivotal role in recognizing early disease onset and initiating timely treatment. Full article