Application Of Metagenomic Next-generation Sequencing Research Articles

Application of metagenomic next-generation sequencing in treatment guidance for deep neck space abscess

BackgroundInfectious etiologies of deep neck space abscess (DNSA) by conventional culture tests can be challenging, which also leads to frequent irrational antibiotic usage. Metagenomic next-generation sequencing (mNGS), as a novel method for analyzing the complex microbial ecosystem from clinical samples, has been utilized in clinical research and practice of various infectious diseases but deep neck space abscess. We here aimed to explore the clinical value of mNGS for pathogen detection and treatment guidance in DNSA patients compared with conventional culture tests.MethodsOne hundred six patients diagnosed with DNSA were retrospectively enrolled and allocated into mNGS group and culture group according to whether mNGS was conducted. The pathogen detection effectiveness was of mNGS was compared with conventional culture. Effectiveness of mNGS-modified antimicrobial therapy was evaluated by comparing the treatment outcomes between two groups.ResultsmNGS showed a significantly higher detection rate than conventional culture (p < 0.05) with faster result acquisition. Treatment success rate of patients in the mNGS group was significantly higher than in the culture group (RR: 1.22, 95%CI: 1.07–1.82, p = 0.033). Besides, patients in the mNGS group had shorter duration of irrational antimicrobial therapy, shorter hospital stay and less medical costs (p < 0.05).ConclusionsmNGS is an effective technology for facilitating pathogen detection and improving treatment outcomes of DNSA patients.

Clinical application of metagenomic next-generation sequencing in rapid diagnosis and prognostic assessment of herpes simplex encephalitis.

Herpes simplex encephalitis (HSE) ranks among the most common causes of severe viral encephalitis. It leads to meningitis or encephalitis, with patients frequently encountering adverse outcomes. In this study, we utilized metagenomic next-generation sequencing (mNGS) to rapidly and accurately detect and identify the HSV pathogen directly from cerebrospinal fluid (CSF) samples, aiming to achieve a definitive diagnosis for encephalitis patients. From 2018 to 2023, we prospectively identified and enrolled 28 patients diagnosed with HSE at Hengshui People's Hospital. CSF samples were subjected to mNGS to facilitate the diagnosis and characterization of HSE in this cohort. We compiled the clinical characteristics, supplementary examinations, and outcomes of HSE patients, with prognosis assessed using the Glasgow Outcome Scale (GOS) scores at discharge, 1 month post-discharge, and 3 months thereafter. In this cohort of 28 patients, 12 were females and 16 males, with a mean age of 41.82 ± 18.23. HSE manifested with a variety of clinical symptoms, the most prevalent being headaches (67.9%), fever exceeding 38°C (60.7%), and altered consciousness (60.7%). Seizures (42.9%), vomiting (35.7%), and speech deficits (35.7%) were frequently observed, with a minority of patients displaying personality changes (28.6%). CSF analysis revealed pleocytosis and a mild increase in protein levels. Magnetic resonance imaging (MRI) abnormalities (28.6%) were primarily confined to the frontal and temporal lobes as well as limbic regions, with no indications of cerebral hemorrhage. Half of the patients exhibited Electroencephalogram (EEG) changes suggestive of encephalitis. HSE was confirmed through mNGS analysis of CSF within 3 days of admission. All patients received empirical treatment with ganciclovir, with 46.4% undergoing hormonotherapy and 32.1% receiving immunoglobulin therapy. At the three-month follow-up, 32.1% had GOS scores <5. HSE often presents with nonspecific signs of encephalitis, and it's not easy for traditional CNS examinations to confirm the diagnosis. mNGS serves as a cutting-edge diagnostic tool for the rapid and precise identification of HSE, facilitating timely clinical diagnosis and intervention to prevent the progression of the disease.

Application of metagenomic next-generation sequencing in the diagnosis of pathogens in patients with diabetes complicated by community-acquired pneumonia.

To explore the clinical utility and optimal timing of metagenomic next-generation sequencing (mNGS) in diagnosing pathogens in patients with diabetes complicated by community-acquired pneumonia (CAP). The study included 50 hospitalized patients diagnosed with diabetes complicated by CAP who underwent conventional microbiological testing (CMT) and mNGS using bronchoalveolar lavage fluid. Among the 50 cases, 16% presented no respiratory symptoms. There were significant increases in inflammatory markers such as C-reactive protein, erythrocyte sedimentation rate, and interleukin-6, with patchy imaging changes being the most prevalent. The positive rates for pathogen detection by mNGS and CMTs were 78 and 21% (P < 0.05). The mNGS was significantly better than the CMTs in the detection of rare pathogens such as Anaerobes, Chlamydia psittaci, Legionella pneumophila, Mycobacterium bovis, Aspergillus fumigatus, and Pneumocystis japonicus (P < 0.05). After clinical interpretation, 85% (22/26) of viruses, 24% (9/37) of bacteria, and 25% (2/8) of fungi were non-pathogen organisms by mNGS. There was a significant difference in the rates of adjustment in anti-infection treatment strategies based on the pathogen detection results from CMTs and mNGS, which were 2 and 46%, respectively (P < 0.05). We found that mNGS was superior to CMTs in terms of the positive rate of pathogen detection, detecting mixed infection incidence, rare pathogen detection rates, and the adjustment of treatment strategies. However, mNGS results need to be interpreted in the context of the clinic.

Comparison of mNGS with conventional methods for diagnosis of cryptococcal meningitis: a retrospective study

The application of metagenomic next-generation sequencing (mNGS) in the diagnosis of cryptococcal meningitis is relatively under characterized. Here, we retrospectively evaluated data from cryptococcal meningitis patients who were tested using mNGS and/or routine testing, including fungal culture, India ink staining, and cryptococcal antigen (CrAg) testing. The performance of mNGS was then assessed. Initial cerebrospinal fluid (CSF) samples were collected from 65 patients with suspected central nervous system (CNS) infection and tested using conventional tests and/or mNGS. mNGS offers a culture-independent approach, facilitating a rapid and unbiased detection of a broad spectrum of pathogens. Patients with bacterial tuberculous or viral meningitis were used as mNGS-positive controls and one autoimmune encephalitis patient was used as an mNGS-negative control. In the 45 patients diagnosed with cryptococcal meningitis, the sensitivity, specificity, positive predictive value, negative predictive value, and concordance rate of mNGS were 92%, 100%, 100%, 90.9%, and 95.6%, respectively. Compared to conventional methods, the sensitivity of mNGS was slightly lower than CrAg tests (96.7%) but higher than India ink (79.5%) and culturing (63.4%). Of the two negative mNGS cases (2/25, 8.0%), one was positive by India ink staining, culture, and CrAg testing, while the other was positive only by CrAg testing. A combination of mNGS and conventional methods enhanced the detection rate to 100%. Our study demonstrates that both CrAg and mNGS offer excellent diagnostic accuracy for cryptococcal meningitis, and utilizing both tests can enhance clinical assessment and patient management.

Application of metagenomic next-generation sequencing (mNGS) to describe the microbial characteristics of diabetic foot ulcers at a tertiary medical center in South China

BackgroundDiabetic foot ulcers (DFUs) are characterized by dynamic wound microbiome, the timely and accurate identification of pathogens in the clinic is required to initiate precise and individualized treatment. Metagenomic next-generation sequencing (mNGS) has been a useful supplement to routine culture method for the etiological diagnosis of DFUs. In this study, we utilized a routine culture method and mNGS to analyze the same DFU wound samples and the results were compared.MethodsForty samples from patients with DFUs at a tertiary medical center in South China were collected, the microorganisms were identified with mNGS and routine culture method simultaneously.ResultsThe results showed that the positive detection rate of microorganisms in DFUs with mNGS was much higher (95% vs. 60%). Thirteen strains of microorganisms were detected with routine culture method, and seventy-seven strains were detected with mNGS. Staphylococcus aureus was the most common microorganism detected with culture method, while Enterococcus faecalis was the most common microorganism detected with mNGS. The false negative rate of the culture method was 35%, that was, 14 samples with negative results with culture method were found to be positive with mNGS.ConclusionThe mNGS method had a higher positive detection rate and identified a broader spectrum of microorganisms in DFUs, thus, mNGS provided a more comprehensive understanding of the microbiome of DFUs to facilitate the development of timely and optimal treatment.Trial registrationThe study was conducted in accordance with the Declaration of Helsinki and approved by the Ethical Review Committee of the Fujian Medical University Union Hospital (approval number 2021KY054).

Application of metagenomic next-generation sequencing in the diagnosis of post-stroke infections: a case series study using multiple sample types.

Metagenomic high-throughput sequencing (mNGS) represents a powerful tool for detecting nucleic acids from various pathogens, such as bacteria, fungi, viruses and parasites, in clinical samples. Despite its extensively employed in the pathogen diagnosis for various infectious diseases, its application in diagnosing stroke-related infection, and its potential impact on clinical decision-making, anti-infection treatment, clinical intervention, and patient prognosis remain insufficiently explored. Additionally, while mNGS offers promising potential, it facts limitations related to sensitivity, specificity, cost, and standardization, which could influence its integration into routine clinical practice. We retrospectively analyzed 18 stroke patients admitted to the First Affiliated Hospital of Medical College of Shantou University from January to February 2023, comparing culture-based methods with mNGS detection, and assessing its significance in etiological diagnosis. Additionally, we evaluated the performance differences among various sequencing platforms. Among the 18 stroke patients enrolled, pulmonary infections were identified in 7 cases, urinary tract infections in 1 case, central nervous system infections in 10 cases, and combined pulmonary and central nervous system infections in 2 cases, with 2 cases yielding negative results. mNGS detected pathogens in 13 cases, aligning with clinical diagnoses (75% concordance), whereas culture-based methods yielded positive results in only 6 cases (22% concordance). Importantly, for 9 of the 18 patients, adjustments to anti-infective treatment regimens based on mNGS results led to improved symptomatic relief and infection control. This suggests that mNGS can contribute to more timely and precise treatment modifications, particularly for infections with low pathogen loads, potentially enhancing clinical outcomes. Our findings highlights the utility of mNGS in diagnosing stroke-associated infections by providing a more comprehensive etiological diagnosis compared to traditional method. While mNGS shows promise in enhancing diagnostic accuracy and guiding clinical treatment, it high cost and technical challenges need addressing before widespread clinical adoption. Future research should focus on optimizing mNGS protocols, integration it with convertional diagnostic tools, and evaluating its cost-effectiveness and clinical impact through larger, multicentric studies.

The role of metagenomic next-generation sequencing in diagnosing and managing post-kidney transplantation infections.

Kidney transplantation (KT) is a life-saving treatment for patients with end-stage renal disease, but post-transplant infections remain one of the most significant challenges. These infections, caused by a variety of pathogens, can lead to prolonged hospitalization, graft dysfunction, and even mortality, particularly in immunocompromised patients. Traditional diagnostic methods often fail to identify the causative organisms in a timely manner, leading to delays in treatment and poorer patient outcomes. This review explores the application of metagenomic next-generation sequencing (mNGS) in the diagnosis of post-KT infections. mNGS allows for the rapid, comprehensive detection of a wide range of pathogens, including bacteria, viruses, fungi, and parasites, without the need for culture-based techniques. We discuss the advantages of mNGS in early and accurate pathogen identification, its role in improving patient management, and the potential challenges in its clinical implementation. Additionally, we consider the future prospects of mNGS in overcoming current diagnostic limitations and its potential for guiding targeted therapies, particularly in detecting antimicrobial resistance and emerging pathogens. This review emphasizes the promise of mNGS as an essential tool in improving the diagnosis and treatment of infections in KT recipients.

Application of metagenomic next-generation sequencing in pathogen detection for patients with lower respiratory tract infections caused bymultidrug-resistant organisms and analysis ofrelated factors.

The incidence of lower respiratory tract infections (LRTIs) caused by multidrug-resistant organisms (MDRO) has been high in recent years. However, traditional etiological detection methods have not been able to meet the needs for clinical diagnosis and prognosis of LRTIs. The rapid development of metagenomic next-generation sequencing (mNGS) provides new insights for diagnosis and treatment ofLRTIs. We conducted a retrospective study on 95 patients with lower respiratory tract infections caused by MDRO admitted to our respiratory department from January 2022 to December 2023. Thesepatients underwent mNGS testing and conventional culture testing. Additionally, 150 patients without lower respiratory tract infections caused by MDRO during the same period were included as thenon-MDRO group. General information was collected, and Logistic regression analysis was performed to identify risk factors for MDRO infections in patients with lower respiratory tract infections. Our results show that the time to pathogen detection by mNGS was 50.76 ± 1.730 h, that is significantly shorter than 55.53 ± 2.782 h required for conventional culture testing. The pathogen detection rate by mNGS was 89.47% (85/95), higher than the 67.37% (64/95) identified by conventional testing. In terms of pathogen genus distribution, mNGS detected a total of 279 pathogens, while conventional testing detected 121 pathogens. Logistic multivariate regression analysis identified that the use of more thantwo antibiotics, invasive procedures, invasive mechanical ventilation for ≥7 days, and stay in therespiratory intensive care unit (RICU) for ≥7 days were the main influencing factors for lower respiratory tract infections caused by MDRO (P < 0.05).

Clinical Application of Metagenomic Next-Generation Sequencing (mNGS) in Patients with Early Pulmonary Infection After Liver Transplantation

Clinical Application of Metagenomic Next-Generation Sequencing (mNGS) in Patients with Early Pulmonary Infection After Liver Transplantation

Effect of metagenomic next-generation sequencing on clinical outcomes in adults with severe pneumonia post-cardiac surgery: a single-center retrospective study

Reports on the application of metagenomic next-generation sequencing (mNGS) in adult patients with severe pneumonia after cardiac surgery remain limited. This study aimed to evaluate the clinical outcomes of mNGS analysis of bronchoalveolar lavage fluid (BALF) in such patients.A retrospective cohort study was conducted on adult patients with severe pneumonia after cardiac surgery. Samples were collected from patients in the surgical intensive care unit (SICU) of Sichuan Provincial People’s Hospital between January 2019 and March 2024. Upon diagnosis of severe pneumonia, bronchoalveolar lavage fluid was obtained via bronchoscopy within 24 h. The mNGS group was composed of patients tested using mNGS and conventional microbiological tests. BALF was detected only by the conventional microbiological test (CMT) method in the CMT group, which involved examining bacterial and fungal smears and cultures at least. We reviewed a total of 4,064 cardiac surgeries, and based on the inclusion criteria, a total of 113 adult patients with severe pneumonia after cardiac surgery were included in this study. The overall positive rate detected by mNGS was significantly higher than that of the culture method (98% vs. 58%, P<0.0001). After receipt of the microbiological results, the mNGS group exhibited a higher incidence of antibiotic adjustments in comparison to the CMT group (P = 0.0021). After adjusting the treatment plan based on microbial testing results, the mNGS group showed an improvement in ventilator-free days within 28 days (P = 0.0475), with a shorter duration of invasive ventilation compared to the CMT group (P = 0.0208). The detection of mNGS can significantly improve the Acute Physiology and Chronic Health Evaluation-II (APACHE II) score (P = 0.0161) and Sequential Organ Failure Assessment (SOFA) score (P = 0.0076) on the 7th day after admission to the SICU. In this study, the mNGS group showed signs of having a positive impact on the length of stay in ICU (median: 9 days, IQR: 7–10 days vs. median: 10 days, IQR: 8-13.75 days, P = 0.0538), length of stay in Hospital (median: 20 days, IQR: 17–28 days vs. median: 25 days, IQR: 18–29 days, P = 0.1558), mortality in 28 days (19% vs. 20%, P = 0.8794), in-hospital mortality (19% vs. 22%, P = 0.7123); however, statistical analysis did not confirm these differences to be significant. mNGS could serve as a valuable complement to conventional diagnostic approaches in adult patients with severe pneumonia after cardiac surgery, potentially improving diagnostic accuracy and leading to more precise and timely interventions, with significant potential to inform clinical decision-making and enhance patient outcomes.

Application of metagenomic next-generation sequencing in the diagnosis of infectious diseases.

Metagenomic next-generation sequencing (mNGS) is a transformative approach in the diagnosis of infectious diseases, utilizing unbiased high-throughput sequencing to directly detect and characterize microbial genomes from clinical samples. This review comprehensively outlines the fundamental principles, sequencing workflow, and platforms utilized in mNGS technology. The methodological backbone involves shotgun sequencing of total nucleic acids extracted from diverse sample types, enabling simultaneous detection of bacteria, viruses, fungi, and parasites without prior knowledge of the infectious agent. Key advantages of mNGS include its capability to identify rare, novel, or unculturable pathogens, providing a more comprehensive view of microbial communities compared to traditional culture-based methods. Despite these strengths, challenges such as data analysis complexity, high cost, and the need for optimized sample preparation protocols remain significant hurdles. The application of mNGS across various systemic infections highlights its clinical utility. Case studies discussed in this review illustrate its efficacy in diagnosing respiratory tract infections, bloodstream infections, central nervous system infections, gastrointestinal infections, and others. By rapidly identifying pathogens and their genomic characteristics, mNGS facilitates timely and targeted therapeutic interventions, thereby improving patient outcomes and infection control measures. Looking ahead, the future of mNGS in infectious disease diagnostics appears promising. Advances in bioinformatics tools and sequencing technologies are anticipated to streamline data analysis, enhance sensitivity and specificity, and reduce turnaround times. Integration with clinical decision support systems promises to further optimize mNGS utilization in routine clinical practice. In conclusion, mNGS represents a paradigm shift in the field of infectious disease diagnostics, offering unparalleled insights into microbial diversity and pathogenesis. While challenges persist, ongoing technological advancements hold immense potential to consolidate mNGS as a pivotal tool in the armamentarium of modern medicine, empowering clinicians with precise, rapid, and comprehensive pathogen detection capabilities.

Improving the application of metagenomic next-generation sequencing for pathogen diagnosis in infections related to hematological diseases

Patients with hematologic diseases are prone to infections, and infection-related mortality is high in this population. Accurate diagnosis of infectious pathogens is crucial; however, conventional microbiological testing often fails to meet clinical needs, presenting a significant challenge in clinical practice. Metagenomic next-generation sequencing (mNGS), with its high sensitivity, broad coverage, and short turnaround time, has been widely adopted in diagnosing infectious diseases. This article focuses on the application of mNGS technology in diagnosing infectious pathogens in patients with hematologic diseases. It analyzes the challenges encountered in clinical practice and explores future trends in the field. This work aims to provide clinicians with an in-depth understanding of the value and limitations of mNGS in diagnosing infections in hematologic patients, and to promote its rational application.

Application of metagenomic next-generation sequencing with brain tissue biopsy for diagnosing intracranial lesions in people with HIV.

Application of metagenomic next-generation sequencing with brain tissue biopsy for diagnosing intracranial lesions in people with HIV.

The clinical application of metagenomic next-generation sequencing in immunocompromised patients with severe respiratory infections in the ICU.

Early targeted antibiotic therapy is crucial for improving the prognosis of immunocompromised patients with severe respiratory infections (SRIs) in the intensive care unit (ICU). Metagenomic next-generation sequencing (mNGS) has shown significant value in pathogen detection, but research on lower respiratory tract microorganisms remains limited. This study enrolled 234 patients with SRIs in the ICU, and individuals were categorized into immunocompromised and immunocompetent groups. We compared the diagnostic performance of mNGS using bronchoalveolar lavage fluid (BALF) with conventional microbiological tests (CMTs) and analyzed the value of mNGS in immunocompromised patients with SRIs in the ICU. Among all patients, the pathogenic microorganism detection rate of mNGS was higher than that of CMTs (94.02% vs 66.67%, P < 0.05), both in the immunocompromised group (95.0% vs 58.75%, P < 0.05) and the immunocompetent group (93.51% vs 71.43%, P < 0.05). mNGS detected more pathogens than CMTs did (167 vs 51), identifying 116 organisms that were missed by CMTs. The proportion of antibiotic regimen adjustments based on mNGS results was significantly higher compared to CMTs in both the immunocompromised (70.00% vs 17.50%, P < 0.05) and immunocompetent groups (48.70% vs 15.58%, P < 0.05). In the immunocompromised group, patients who had their antibiotic treatment adjusted on mNGS results had improved prognosis, with significantly lower ICU mortality (8.93% vs 50%, P < 0.05) and 28-day mortality rates (30.36% vs 68.75%, P < 0.05) than CMTs. In the immunocompetent group, no statistically significant differences were observed in ICU mortality or 28-day mortality (20.00% vs 33.33%, P > 0.05; 42.67% vs 45.83%, P > 0.05). mNGS shows significant value in detecting pathogens in immunocompromised patients with SRIs in ICU. For immunocompromised patients who respond poorly to empirical treatment, mNGS can provide an etiological basis, helping adjust antibiotic regimens more precisely and thereby improving patient prognosis.

Clinical Application of Metagenomic Next-Generation Sequencing in Sepsis Patients with Early Antibiotic Treatment.

This study aimed to evaluate the clinical utility of metagenomic next-generation sequencing (mNGS) in sepsis patients who received early empirical antibiotic treatment. A retrospective analysis was conducted on clinical data from sepsis patients diagnosed in the Emergency Intensive Care Unit (EICU) between April 2019 and May 2023. All patients underwent standard conventional microbiological testing. Patients were categorized into either the mNGS group or the control group based on whether they underwent mNGS tests. Baseline variables were matched using propensity scores. Out of 461 sepsis patients screened, 130 were included after propensity matching, with 65 patients in each group. Despite prior antibiotic treatment, 57 cases (87.69%) in the mNGS group had positive mNGS results, exceeding the culture detection rate (52.31%). Besides, a higher proportion of patients in the mNGS group experienced antibiotic adjustments compared to the control group (72.31% vs 53.85%). Mortality rates were also compared based on the duration of antibiotic exposure before mNGS sampling. Patients exposed to antibiotics for less than 24hours had a lower mortality rate compared to those exposed for over 8 days (22.22% vs 42.86%). COX multivariate analysis identified mNGS testing, underlying diseases, lymphocyte percentage, infection site (respiratory and bloodstream) as independent risk factors for mortality in sepsis patients. With increased antibiotic exposure time, the positive rate of culture testing significantly decreased (44.44% vs 59.52% vs 35.71%, P = 0.031), whereas the positive rate of mNGS remained stable (77.78% vs 88.10% vs 92.86%, P = 0.557). mNGS demonstrated less susceptibility to antibiotic exposure. Early mNGS detection positively impacted the prognosis of sepsis patients.

Application of metagenomic next-generation sequencing and targeted metagenomic next-generation sequencing in diagnosing pulmonary infections in immunocompetent and immunocompromised patients.

Metagenomic next-generation sequencing (mNGS) technology has been widely used to diagnose various infections. Based on the most common pathogen profiles, targeted mNGS (tNGS) using multiplex PCR has been developed to detect pathogens with predesigned primers in the panel, significantly improving sensitivity and reducing economic burden on patients. However, there are few studies on summarizing pathogen profiles of pulmonary infections in immunocompetent and immunocompromised patients in Jilin Province of China on large scale. From January 2021 to December 2023, bronchoalveolar lavage fluid (BALF) or sputum samples from 546 immunocompetent and immunocompromised patients with suspected community-acquired pneumonia were collected. Pathogen profiles in those patients on whom mNGS was performed were summarized. Additionally, we also evaluated the performance of tNGS in diagnosing pulmonary infections. Combined with results of mNGS and culture, we found that the most common bacterial pathogens were Pseudomonas aeruginosa, Klebsiella pneumoniae, and Acinetobacter baumannii in both immunocompromised and immunocompetent patients with high detection rates of Staphylococcus aureus and Enterococcus faecium, respectively. For fungal pathogens, Pneumocystis jirovecii was commonly detected in patients, while fungal infections in immunocompetent patients were mainly caused by Candida albicans. Most of viral infections in patients were caused by Human betaherpesvirus 5 and Human gammaherpesvirus 4. It is worth noting that, compared with immunocompetent patients (34.9%, 76/218), more mixed infections were found in immunocompromised patients (37.8%, 14/37). Additionally, taking final comprehensive clinical diagnoses as reference standard, total coincidence rate of BALF tNGS (81.4%, 48/59) was much higher than that of BALF mNGS (40.0%, 112/280). Our findings supplemented and classified the pathogen profiles of pulmonary infections in immunocompetent and immunocompromised patients in Jilin Province of China. Most importantly, our findings can accelerate the development and design of tNGS specifically used for regional pulmonary infections.

Metagenomic next-generation sequencing of cerebrospinal fluid reveals etiological and microbiological features in patients with various central nervous system infections.

The application of metagenomic next-generation sequencing (mNGS) in pathogens detection of cerebrospinal fluid (CSF) is limited because clinical, microbiological, and biological information are not well connected. We analyzed the 428 enrolled patients' clinical features, pathogens diagnostic efficiency of mNGS in CSF, microbial community structure and composition in CSF, and correlation of microbial and clinical biomarkers in CSF. General characteristics were unspecific but helpful in formulating a differential diagnosis. CSF mNGS has a higher detection rate (34.6%) compared to traditional methods (5.4%). mNGS detection rate was higher when the time from onset to CSF collection was ≤20 days, the CSF leukocytes count was >200 × 106/L, the CSF protein concentration was >1.3 g/L, or CSF glucose concentration was ≤2.5 mmol/L in non-postoperative bacterial CNS infections (CNSi). CSF was not strictly a sterile environment, and the potential pathogens may contribute to the dysbiosis of CSF microbiome. Furthermore, clinical biomarkers were significantly relevant to CNS pathogens. Clinical data are helpful in choosing a proper opportunity to obtain an accurate result of mNGS, and can speculate whether the mNGS results are correct or not. Our study is a pioneering study exploring the CSF microbiome in different CNSIs.

Application of metagenomic next-generation sequencing in the etiological diagnosis of refractory pneumonia in children.

Metagenomic next-generation sequencing (mNGS) was used to analyze the etiological distribution of refractory pneumonia in children. We compared its efficacy in pathogen diagnosis against traditional methods to provide a basis for clinical adjustment and treatment. A total of 60 children with refractory pneumonia treated at the Department of Respiratory Medicine, Children's Hospital Affiliated with the Capital Institute of Paediatrics, from September 2019 to December 2021 were enrolled in this study. Clinical data (including sex, age, laboratory tests, complications, and discharge diagnosis) and lower respiratory tract specimens were collected, including bronchoalveolar lavage fluid (BALF), deep sputum, pleural effusion, lung abscess puncture fluid, traditional respiratory pathogens (culture, acid-fast staining, polymerase chain reaction, serological testing, etc.), and mNGS detection methods were used to determine the distribution of pathogens in children with refractory pneumonia and to compare the positive rate and diagnostic efficiency of mNGS and traditional pathogen detection for different types of pathogens. Among the 60 children with refractory pneumonia, 43 specimens were positive by mNGS, and 67 strains of pathogens were detected, including 20.90% (14 strains) of which were Mycoplasma pneumoniae, 11.94% (8 strains) were Streptococcus pneumoniae, 7.46% (5 strains) were cytomegalovirus, and 5.97% (4 strains) were Candida albicans. Thirty-nine strains of Mycoplasma pneumoniae (41.03%, 16 strains), Streptococcus pneumoniae (10.26%, 4 strains), Candida albicans (7.69%, 3 strains), and Aspergillus (5.13%, 2 strains) were detected using traditional methods. The positive rate of mNGS detection was 90.48%, and the positive rate of the traditional method was 61.90% (p = 0.050), especially for G+ bacteria. The positive rate of mNGS was greater than that of traditional methods (p < 0.05), but they had no significant difference in detecting G- bacteria, viruses, fungi, or Mycoplasma/Chlamydia. Among the 60 patients, 21 had mixed infections, 25 had single infections, and the other 14 had unknown pathogens. Mycoplasma pneumoniae was most common in both mixed infections and single infections. The sensitivity, specificity, positive predictive value, and negative predictive value of mNGS were 95.45, 37.50, 80.77, and 75.00%, respectively. The sensitivity, specificity, positive predictive value, and negative predictive value of the traditional methods were 72.72, 62.50, 84.21, and 45.45%, respectively. The clinical compliance of mNGS was 80.00%, and that of the traditional method was 70.00%. The sensitivity and negative predictive value of mNGS were high, and the difference in the sensitivity for detecting G+ bacteria was statistically significant (p < 0.05). However, the differences in G- bacteria, fungi, and Mycoplasma/Chlamydia were not statistically significant (p > 0.05). Due to the small sample size, statistical analysis could not be conducted on viral infections. mNGS has higher overall efficacy than traditional methods for the etiological diagnosis of refractory pneumonia in children. The application of mNGS can significantly improve the detection rate of pathogens in children with refractory pneumonia. The sensitivity and negative predictive value of mNGS for detecting G+ bacteria are greater than those of other methods, and it can exclude the original suspected pathogenic bacteria. Unnecessary antibiotic use was reduced, but there was no statistically significant difference in G- bacteria, fungi, or Mycoplasma/Chlamydia.

Application of metagenomic next-generation sequencing for rapid molecular identification in spinal infection diagnosis.

This study aimed to determine the sensitivity and specificity of metagenomic next-generation sequencing (mNGS) for detecting pathogens in spinal infections and to identify the differences in the diagnostic performance between mNGS and targeted next-generation sequencing (tNGS). A total of 76 consecutive patients with suspected spinal infections who underwent mNGS, culture, and histopathological examinations were retrospectively studied. The final diagnosis of the patient was determined by combining the clinical treatment results, pathological examinations, imaging changes and laboratory indicators. The sensitivity and specificity of mNGS and culture were determined. The difference between the two detection rates was statistically significant (p < 0.001), with mNGS exhibiting a significantly higher detection rate (77.6% versus 18.4%). The average diagnosis time of mNGS was significantly shorter than that of bacterial culture (p < 0.001, 1.65 versus 3.07 days). The sensitivity and accuracy of mNGS were significantly higher than that of the culture group (p < 0.001, 82.3% versus 17.5%; 75% versus 27.6%), whereas the specificity of mNGS (42.9%) was lower than that of the culture group (p > 0.05, 42.9% versus 76.9%). The sensitivity, specificity, accuracy, and positive predictive value (PPV) of pus were higher than those of tissue samples for mNGS, whereas for culture, the sensitivity, specificity, accuracy, and PPV of tissue samples were higher than those of pus. tNGS demonstrated higher sensitivity and accuracy in diagnosing tuberculosis (TB) than mNGS (80% versus 50%; 87.5% versus 68.8%). mNGS for spinal infection demonstrated better diagnostic value in developing an antibiotic regimen earlier, and it is recommended to prioritize pus samples for testing through mNGS. Moreover, tNGS outperformed other methods for diagnosing spinal TB and identifying antibiotic-resistance genes in drug-resistant TB.

Application of metagenomic next-generation sequencing in optimizing the diagnosis of ascitic infection in patients with liver cirrhosis

BackgroundMetagenomic next-generation sequencing (mNGS) is an emerging technique for the clinical diagnosis of infectious disease that has rarely been used for the diagnosis of ascites infection in patients with cirrhosis. This study compared mNGS detection with conventional culture methods for the on etiological diagnosis of cirrhotic ascites and evaluated the clinical effect of mNGS.MethodsA total of 109 patients with ascites due to cirrhosis were included in the study. We compared mNGS with conventional culture detection by analyzing the diagnostic results, pathogen species and clinical effects. The influence of mNGS on the diagnosis and management of ascites infection in patients with cirrhosis was also evaluated.ResultsAscites cases were classified into three types: spontaneous bacterial peritonitis (SBP) (16/109, 14.7%), bacterascites (21/109, 19.3%) and sterile ascites (72/109, 66.1%). In addition, 109 patients were assigned to the ascites mNGS-positive group (80/109, 73.4%) or ascites mNGS-negative group (29/109, 26.6%). The percentage of positive mNGS results was significantly greater than that of traditional methods (73.4% vs. 28.4%, P < 0.001). mNGS detected 43 strains of bacteria, 9 strains of fungi and 8 strains of viruses. Fourteen bacterial strains and 3 fungal strains were detected via culture methods. Mycobacteria, viruses, and pneumocystis were detected only by the mNGS method. The mNGS assay produced a greater polymicrobial infection rate than the culture method (55% vs. 16%). Considering the polymorphonuclear neutrophil (PMN) counts, the overall percentage of pathogens detected by the two methods was comparable, with 87.5% (14/16) in the PMN ≥ 250/mm3 group and 72.0% (67/93) in the PMN < 250/mm3 group (P > 0.05). Based on the ascites PMN counts combined with the mNGS assay, 72 patients (66.1%) were diagnosed with ascitic fluid infection (AFI) (including SBP and bacterascites), whereas based on the ascites PMN counts combined with the culture assay, 37 patients (33.9%) were diagnosed with AFI (P < 0.05). In 60 (55.0%) patients, the mNGS assay produced positive clinical effects; 40 (85.7%) patients had their treatment regimen adjusted, and 48 patients were improved. The coincidence rate of the mNGS results and clinical findings was 75.0% (60/80).ConclusionsCompared with conventional culture methods, mNGS can improve the detection rate of ascites pathogens, including bacteria, viruses, and fungi, and has significant advantages in the diagnosis of rare pathogens and pathogens that are difficult to culture; moreover, mNGS may be an effective method for improving the diagnosis of ascites infection in patients with cirrhosis, guiding early antibiotic therapy, and for reducing complications related to abdominal infection. In addition, explaining mNGS results will be challenging, especially for guiding the treatment of infectious diseases.