Metagenomic Next-generation Sequencing Research Articles

Nonunions and fracture-related infections represent a significant complication in orthopedic and trauma care, with their incidence rising due to an aging, more comorbid global population and the escalating threat of multi-resistant pathogens. This narrative review highlights pivotal advancements in diagnostics and therapeutic approaches, while also providing an outlook on future directions. Diagnostic methodologies have significantly evolved from traditional cultures to sophisticated molecular techniques like metagenomic next-generation sequencing and advanced imaging. Simultaneously, therapeutic strategies have undergone substantial refinement, encompassing orthoplastic management for infected open fractures and the innovative application of antibiotic-loaded bone substitutes for local drug delivery. The effective integration of these possibilities into daily patient care critically depends on specialized centers. These institutions play an indispensable role in managing complex cases and fostering innovation. Despite considerable progress over the past 25 years, ongoing research, interdisciplinary collaboration, and a steadfast commitment to evidence-based practice remain crucial to transforming management for the future.

Clinical characterization and diagnosis of 14 patients with Chlamydia psittaci pneumonia.

The diagnostic efficacy of metagenomic next-generation sequencing in spontaneous bacterial peritonitis: System review and meta-analysis

This retrospective study compared metagenomic next-generation sequencing (mNGS) and traditional culture for pathogen detection in 43 patients with lower respiratory tract infections (LRTI), including 34 COVID-19 cases (14 critical, 20 non-critical) and 9 non-COVID controls. mNGS demonstrated superior sensitivity (95.35% vs. 81.08%) and broader pathogen coverage, identifying 36.36% of bacteria and 74.07% of fungi detected by cultures. Concordance between methods was observed in 63% of cases. Severe COVID-19 patients exhibited reduced respiratory microbiota abundance, potentially linked to viral dominance or therapeutic interventions. Clinical outcomes correlated positively with inflammatory markers (procalcitonin/PCT, N-terminal pro-B-type natriuretic peptide/N-proBNP, neutrophils, lactate dehydrogenase/LDH,neutrophil-to-lymphocyte ratio/NLR) and negatively with lymphocytes, highlighting systemic inflammation's role in disease progression. While mNGS offers rapid, high-sensitivity pathogen profiling, limitations include small sample sizes, unresolved specificity concerns and unmeasured confounders. The study underscores mNGS as a promising tool for LRTI diagnosis in COVID-19, though larger prospective cohorts and standardized outcome metrics are needed to validate clinical utility, optimize interpretation, and address cost-effectiveness compared to conventional methods.

Rapid and accurate identification of pathogens is essential for managing lung infections in patients following kidney transplantation. This study aimed to compare the diagnostic performance and clinical utility of conventional detection methods and metagenomic next-generation sequencing (NGS) in kidney transplant recipients with respiratory infections. We conducted a retrospective analysis of metagenomic NGS and conventional detection method results in 71 patients, examining the spectrum of pathogen detection characteristics between the 2 methods. The overall positivity rate of conventional detection methods was statistically significantly lower than that of metagenomic NGS (61.97% vs 84.51%, P = .004). Among the 38 participants who tested positive by both methods, metagenomic NGS identified a greater number of pathogens than conventional detection methods. Following metagenomic NGS results, antibiotic therapy was modified in 71.83% of participants, leading to improved prognoses in 33.33% of patients. In additionally, metagenomic NGS demonstrated a shorter turnaround time than conventional detection methods. The most prevalent bacteria identified in pulmonary infections among kidney transplant recipients were Klebsiella pneumoniae, while cytomegalovirus was the most common virus and Pneumocystis jirovecii was the predominant fungus. This study offers preliminary insights into the spectrum of pathogens responsible for pulmonary infections following kidney transplantation, laying the foundation for better understanding their clinical characteristics. In patients with post-transplant pulmonary infections, metagenomic NGS outperforms conventional detection methods in terms of pathogen detection, speed, positivity rate, sensitivity, and ability to diagnose mixed infections.

Background Early and accurate pathogen identification is crucial for managing central nervous system infections (CNSIs). While Metagenomic Next-Generation Sequencing (mNGS) offers rapid and sensitive pathogen detection, its cost-effectiveness in postoperative neurosurgical patients in critical care settings remains underexplored. Our study aims to investigate the clinical health economic value of mNGS in detecting pathogens of CNSIs after neurosurgery. Methods In this prospective pilot study, 60 patients with CNSIs at Beijing Tiantan Hospital ICU (March 2023-January 2024) were randomized 1:1 to mNGS or conventional pathogen culture groups. A decision-tree model compared cost-effectiveness using incremental cost-effectiveness ratios (ICERs). A decision-tree model was used to compare the cost-effectiveness between mNGS and traditional pathogen culture methods using incremental cost-effectiveness ratios (ICERs). Results From March 2023 to January 2024, 60 patients were included. mNGS demonstrated superior diagnostic efficiency with shorter turnaround time (1 vs 5 days; _P_<0.001) and lower anti-infective costs (¥18,000 vs ¥23,000; _P_=0.02). Despite higher detection costs (¥4,000 vs ¥2,000; _P_<0.001), the ICER of ¥36,700 per additional timely diagnosis suggested cost-effectiveness at China’s GDP-based WTP threshold. No significant differences in hospitalization duration or total costs were observed (_P_>0.05). Conclusion mNGS improves diagnostic efficiency and reduces antimicrobial expenditure for postoperative CNSIs in critical care, demonstrating favorable cost-effectiveness when considering clinical outcome gains.

Metagenomic next-generation sequencing (mNGS) guides targeted antibiotic therapy and reduces mortality in sepsis: a propensity-matched retrospective cohort study

Objective This study aimed to compare the diagnostic performance of conventional microbial culture and metagenomic next-generation sequencing (mNGS) in detecting pathogens in periprosthetic joint infection (PJI) and to identify factors contributing to discrepancies between these two methods. Methods A total of 167 patients with suspected PJI (including PJI patients and aseptic failure patients) who underwent revision joint replacement at our center from September 2017 to April 2024 were enrolled. Demographic data, prior antibiotic use, and results of microbial culture and mNGS were documented. Joint fluid, periprosthetic tissue, or prosthetic ultrasonic fluid samples were collected, and at least one sample from each patient underwent both microbial culture and mNGS testing. In the light of the concordance between culture and mNGS results, patients were divided into the detection consistent and detection inconsistent groups. The differences in pathogen detection between the two models were compared, and factors contributing to discordant results were analyzed. Results The prior antibiotic use (OR = 2.137, 95% CI = 1.069-4.272, P = 0.032), polymicrobial infections (OR = 3.245, 95% CI = 1.278-8.243, P = 0.013), infection caused by rare pathogens (OR = 2.735, 95% CI = 1.129-6.627, P = 0.026), and intraoperative tissue specimens (OR = 2.837, 95% CI = 1.007-7.994, P = 0.049) were identified as risk factors for discordance between microbial culture and mNGS results, particularly in cases with negative microbial culture but positive mNGS findings. Conversely, consistency in specimen type (OR = 0.471, 95%CI=0.254-0.875, P = 0.017) was identified as a protective factor against discordance. Conclusion Clinicians should optimize diagnostic strategies by tailoring microbial culture methods to the patient’s clinical condition and integrating mNGS testing where appropriate. It is recommended to use tissue specimens from the same anatomical site across multiple tests while sampling from different regions when necessary. Although this approach may increase costs, it significantly enhances the accuracy of pathogen identification and facilitates more effective treatment.

This study explored the metagenomic sequencing methodology for analyzing the breast milk microbiome and elucidated its composition. Twenty-two breast milk samples were collected from 11 healthy lactating women. By optimizing microbial cell wall disruption parameters and developing a nucleic acid extraction method, microbial DNA/RNA libraries were constructed and subjected to metagenomic next-generation sequencing (mNGS), microbial standards spiked into breast milk at serial dilutions served to validate the method's reliability. The sequencing data underwent rigorous quality control and classification using the Kraken2 software and a self-generated database. The breast milk microbiome was found to comprise 21 phyla, 234 genera, and 487 species, with Firmicutes and Proteobacteria being the dominant phyla. At the genus level, Staphylococcus and Streptococcus were the most abundant, while at the species level, Staphylococcus aureus, Streptococcus bradystis, and Staphylococcus epidermidis were the most prevalent. The microbial profiles of the left and right breast milk samples were consistent at the phylum, genus, and species levels. Besides common bacteria, diverse viral, eukaryotic, and archaeal sequences were also detected. Functional profiling revealed that the "lactose and galactose degradation I" pathway accumulated the highest read count, whereas the L-valine biosynthesis pathway was detected most frequently. This study provides a comprehensive understanding of the healthy breast milk microbiome, highlighting the presence of specific flora colonization and the distinct yet correlated microbial environments in bilateral breast milk, laying the groundwork for future research into the interactions between breast milk microbiota and maternal and infant health outcomes.

Molecular tests without well-defined test performance characteristics are increasingly available for diagnosis of infectious diseases. These tests present a diagnostic stewardship challenge for institutions. We share the results of a local modified Delphi consensus undertaken to define appropriate scenarios for use of plasma metagenomic next-generation sequencing.

To thoroughly describe unique intraocular infections diagnosed by metagenomic next-generation sequencing (mNGS). A retrospective case series of patients presenting with challenging atypical intraocular infections at Tel Aviv Sourasky Medical Center during 2024. Clinical and demographic data, as well as mNGS results were extracted from patient records. mNGS was performed on the Illumina NextSeq500 platform using a custom bioinformatics pipeline. The following parameters were examined: Reads Per Million, Reads Per Million-ratio to negative control and E-index (K-mers*coverage/reads). The study included three patients with novel presentations of intraocular infections, manifesting with atypical clinical manifestations and negative routine diagnostic workups. mNGS allowed the identification of Cytomegalovirus in a 43-year-old male with a history of autosomal dominant hyper-IgE syndrome, Bartonella henselae infection manifesting with photoreceptoritis, retinal vasculitis and global retinal dysfunction in a healthy 28-year-old female, and polymicrobial endophthalmitis with Rothia mucilaginosa and Pantoea agglomerans following intravitreal faricimab injection for neovascular age-related macular degeneration in an 81-year-old male. Treatment regimens were adjusted based on mNGS results. Metagenomic next-generation sequencing has an important role in the diagnosis of challenging intraocular infections. It enables comprehensive pathogen identification and enhances the precision of treatment strategies.

Background The early diagnosis of pulmonary cryptococcosis (PC) remains challenging due to the low sensitivity and prolonged turnaround time of conventional diagnostic methods. Despite the broad-spectrum pathogen detection capability of metagenomic next-generation sequencing (mNGS), its clinical utility in the diagnosis and therapeutic management of pulmonary cryptococcosis remains underexplored. Methods In this retrospective study, 31 patients diagnosed with Cryptococcus infection through mNGS at The First Affiliated Hospital of Zhengzhou University between July 2023 to March 2025 were included. data on clinical characteristics, treatment regimens, and patient prognosis were systematically collected. Results Compared to conventional pathogen detection methods, mNGS demonstrated superior sensitivity, shorter turnaround time (1.00 d vs. 4.50 d, p = 0.002), and significantly reduced interval from admission to clinical decision-making (3.50 d vs. 9.00 d, p = 0.002). Among 31 patients with mNGS-identified cryptococcal infection, only 12 underwent fungal culture, with merely 1 case yielding positive results (positivity rate: 8.33%). Antimicrobial therapy was optimized for all patients based on mNGS findings. During post-discharge follow-up of 27 cases, 1 patient experienced disease recurrence, 1 died from tumor metastasis, and 1 was lost to follow-up. Conclusion Our retrospective analysis revealed that mNGS facilitated treatment optimization, improved clinical outcomes, and provided crucial evidence supporting the precision management of pulmonary cryptococcosis.

This article reports a rare case of sternal osteomyelitis caused by Coxiella burnetii. The patient, a 59-year-old male with a history of aortic dissection and hypertension, had undergone ascending aorta replacement and aortic valvuloplasty. He was admitted in May 2022 due to a chest mass that had been enlarging since its discovery in October 2021. Despite antibiotic treatment, the symptoms did not improve, and he experienced recurrent low-grade fevers. Upon admission, CT imaging revealed multiple mediastinal lymph node enlargements and a cystic shadow anterior to the sternum handle. The infection was confirmed as Coxiella burnetii through surgical debridement and metagenomic next-generation sequencing (mNGS). The treatment was subsequently changed to doxycycline and hydroxychloroquine. Positron emission computed tomography with 18F-fluorodeoxyglucose ([18F] FDG PET/CT) imaging showed that the infection was localized to the sternum, excluding vascular and implant infections. The patient completed an 18-month course of antibiotics, leading to controlled infection and normalized renal function. This case highlights the complexities of diagnosing and managing Q fever osteomyelitis in patients post-cardiovascular surgery, demonstrating the crucial roles of mNGS and [18F] FDG PET/CT in rapid diagnosis and effective treatment. These findings provide valuable insights and guidance for managing similar cases in the future.

Application of metagenomic next-generation sequencing in the precise and rapid diagnosis of spinal infections.

Following recognition that our hospital had higher use of plasma metagenomic next-generation sequencing than our peers, we implemented a process for approval by infectious diseases before test collection. This intervention is calculated to result in a direct cost savings of $79,505-$84,057/year, driven mainly by reduced laboratory costs.

Infected pancreatic necrosis (IPN) is a severe complication of acute pancreatitis, requiring prompt diagnosis. Conventional microbial culture, the current gold standard, has limitations in sensitivity and turnaround time. Metagenomic next-generation sequencing (mNGS) offers rapid, comprehensive pathogen detection, but its diagnostic performance for IPN remains unclear. We conducted a systematic review and meta-analysis following PRISMA-DTA guidelines, prospectively registered in PROSPERO (CRD420251008574). PubMed, Embase, and Web of Science databases were searched from inception to March 2025. Seven studies (313 patients) evaluating mNGS for IPN diagnosis were included, with four providing direct comparisons to culture. Pooled sensitivity, specificity, and area under the curve (AUC) were calculated using a random-effects model. Heterogeneity was assessed using I2 statistics. In double-arm analysis, mNGS showed significantly higher sensitivity (0.87, 95% CI: 0.72-0.95) than culture (0.36, 95% CI: 0.23-0.51), with comparable specificity (0.83 for both). The AUC for mNGS (0.92, 95% CI: 0.79-0.94) surpassed that of culture (0.52, 95% CI: 0.27-0.86). Single-arm analysis confirmed mNGS as a reliable standalone test (sensitivity: 0.86; specificity: 0.85; AUC: 0.89). A threshold effect (r = -0.991) indicated variability in diagnostic criteria across studies. mNGS outperforms culture in diagnosing IPN, offering higher sensitivity and faster results. Its ability to detect diverse pathogens, including fastidious and polymicrobial infections, makes it a valuable tool for early intervention. However, challenges like cost, standardization, and interpretation persist. Future studies should focus on prospective validation and cost-effectiveness to integrate mNGS into routine clinical practice.

We report the case of a 59-year-old HIV-negative male diagnosed with a brain abscess following vasculitis-associated stroke. The causative pathogen remains unidentified using conventional methods including culture and rapid multiplex PCR. Craniotomy and biopsy were performed to establish a definitive diagnosis, and metagenomic next-generation sequencing (mNGS) of the abscess tissue identified Candida tropicalis as the causative pathogen. This case highlights the utility of mNGS in identifying pathogens in culture-negative CNS infections, even when conventional methods fail to detect the causative agent, particularly when evaluating abscess pus.

Diagnosis of spinal infections caused by fastidious bacteria: a multicenter, retrospective observational study.

Development and evaluation of CD45-conjugated magnetic particles-based host cell depletion for enhanced metagenomic next-generation sequencing in bloodstream infection.

BackgroundListeria monocytogenes meningoencephalitis (LMM) is a rare but severe central nervous system (CNS) infection. This study aimed to characterize the clinical manifestations, diagnostic findings, treatment responses, and prognostic factors associated with LMM.MethodsWe retrospectively analyzed the clinical data of 13 patients diagnosed with LMM at Xuzhou Medical University Affiliated Hospital between 2018 and 2023. An additional five cases were identified through a literature search in the China National Knowledge Infrastructure (CNKI) and Wanfang databases from 2019 to 2022. Clinical features, cerebrospinal fluid (CSF) and blood test results, imaging findings, treatments, and outcomes were summarized.ResultsAmong 18 patients (11 males, 7 females; mean age 51.6 ± 17.2 years), all had acute onset with fever (100%), headache (83.3%), and altered consciousness (77.8%). CSF analysis showed elevated pressure in 83.3%, increased leukocytes and protein in all cases, and variable glucose levels. LM was cultured from CSF in 10 patients and detected via metagenomic next-generation sequencing (mNGS) in 12. Imaging findings included hydrocephalus in 4 cases and meningeal enhancement in 2 cases. Full recovery was observed in 2 cases, improvement in 9, palliative care in 3, and death in 4. Early seizures and hydrocephalus were linked to worse outcomes.ConclusionListeria monocytogenes meningoencephalitis is a rapidly progressive CNS infection with variable presentations and significant risk of poor outcomes. Early recognition, appropriate antimicrobial coverage, and timely diagnostic testing are essential to improving prognosis.Trial registrationNot applicable.