Metagenomic Next-generation Sequencing Research Articles

Primary nasopharyngeal carcinoma with Sjögren's syndrome and positive cerebrospinal fluid Epstein-Barr virus: a case report and literature review

The study presents an analysis of the diagnostic and treatment protocol for a patient with a first episode of nasopharyngeal carcinoma who also has Sjogren's syndrome and Epstein-Barr Virus (EBV) positive cerebrospinal fluid, as detected through metagenomic next-generation sequencing (mNGS). It reviews existing literature to examine the connections between EBV and various conditions including Sjogren's syndrome, encephalitis or meningitis, and nasopharyngeal carcinoma, emphasizing the importance of EBV positive cerebrospinal fluid. The study focuses on a case from the Eighth Medical Center of the General Hospital of the People's Liberation Army, where a patient was admitted with headaches as the primary symptom on March 3, 2021. This patient had a history of Sjogren's syndrome and was later diagnosed with nasopharyngeal carcinoma. The research involved reviewing both domestic and international databases for cases related to cerebrospinal fluid EBV positive encephalitis or meningitis, and nasopharyngeal carcinoma. It aimed to aggregate data on demographics, initial symptoms, treatment methods, and patient outcomes. Findings suggest that positive cerebrospinal fluid EBV is linked to autoimmune diseases, viral encephalitis or meningitis, and nasopharyngeal carcinoma, albeit infrequently in the context of Sjogren's syndrome. Notably, EBV positive cerebrospinal fluid is commonly associated with recurrent nasopharyngeal carcinoma rather than initial episodes. The study concludes that for patients with an immune condition, exhibiting symptoms like headaches or cranial nerve issues, or in cases where nasopharyngeal carcinoma is suspected, early testing through cerebrospinal fluid mNGS or EBV DNA is recommended. This approach facilitates risk assessment, prognosis determination, and the creation of individualized treatment plans.

Diagnostic value of DNA or RNA-based metagenomic next-generation sequencing in lower respiratory tract infections

ObjectivesWe aimed to evaluate and compare the diagnostic performance of RNA-mNGS and DNA-mNGS workflow in bacterial pneumonia, fungal pneumonia and tuberculosis. MethodsA total of 134 cases suspected pneumonia undergoing both DNA and RNA based mNGS of bronchoalveolar lavage fluid (BALF) and also traditional etiological examination were evaluated retrospectively.Sensitivity, specificity, PPV, NPV and accuracy rate of DNA and RNA based mNGS were estimated. ResultsIn the diagnosis performance of bacterial pathogens in LRTIs,the specificity of RNA-mNGS was higher than that of DNA-mNGS(82.3 % vs. 61.9 %, P < 0.01). There was no significant difference of sensitivity between the two process(71.4 % vs. 85.7 %, P = 0.375).In the diagnosis performance of fungal pathogens in LRTIs,the specificity of RNA-mNGS was higher than that of DNA-mNGS (72.3 % vs. 27.3 %,p < 0.001). There was no significant difference of sensitivity between the two process(96.5 % vs. 98.8 %,p = 0.125).In the diagnosis performance of tuberculosis in LRTIs,the sensitivity of DNA-mNGS was higher than that of RNA-mNGS (91.7 % vs. 33.3 %,p = 0.016),the specificity was similar in the two process (100 %). ConclusionsRNA-mNGS may reduced the misdiagnosis rate of bacterial and fungal pathogens in LRTIs.Compared to RNA-mNGS, DNA-mNGS may could improve the diagnostic rate of tuberculosis.

Diagnostic Role of Metagenomic Next-Generation Sequencing in Tubercular Orthopedic Implant-Associated Infection.

The diagnosis of tubercular orthopedic implant-associated infection (TB-IAI) is challenging. This study evaluated the value of metagenomic next-generation sequencing (mNGS) for the diagnosis of TB-IAI and developed a standardized diagnostic procedure for TB-IAI. The records of all patients with TB-IAI diagnosed and treated at our institution between December 2018 and September 2022 were retrospectively reviewed. Patient demographic characteristics, medical history, laboratory test, microbial culture, histopathology, and mNGS results, and time to diagnosis were recorded. The diagnostic efficiency of mNGS for TB-IAI was assessed by comparing the results and diagnostic time with that of other diagnostic modalities. Ten patients were included in the analysis, including eight with prosthetic joint infections and two with fracture-related infections. The mNGS positivity rate was 100% (10/10), which was higher than that of TB-antibody (11%, 1/9), real-time quantitative polymerase chain reaction (22%, 2/9), T-SPOT.TB (25%, 2/8), purified protein derivative (50%, 4/8), microbial culture (50%, 5/10), and histopathology (20%, 2/10). mNGS shortened the time to diagnosis of TB-IAI. A standardized diagnostic procedure for TB-IAI was developed based on the findings. mNGS is useful for the diagnosis of TB-IAI. mNGS is recommended in cases where it is difficult to identify a pathogen using routine diagnostic tests. The standardized diagnostic procedure might improve TB-IAI diagnosis. TB-IAI is a rare infection, which occurs after orthopedic surgery and hard to diagnose microbiologically. mNGS is a new detection technique not yet discussed in current literature as a means for TB-IAI diagnostics. Here we describe a cohort of patients with TB-IAI diagnosed by mNGS show high efficiency of mNGS for detection of this pathology and present a clinical algorithm supplementing conventional methods for TB-IAI assessment.

High Detection Frequency of Vaccine-Associated Polioviruses and Non-Polio Enteroviruses in the Stools of Asymptomatic Infants from the Free State Province, South Africa.

Enterovirus (EV) infections are widespread and associated with a range of clinical conditions, from encephalitis to meningitis, gastroenteritis, and acute flaccid paralysis. Knowledge about the circulation of EVs in neonatal age and early infancy is scarce, especially in Africa. This study aimed to unveil the frequency and diversity of EVs circulating in apparently healthy newborns from the Free State Province, South Africa (SA). For this purpose, longitudinally collected faecal specimens (May 2021-February 2022) from a cohort of 17 asymptomatic infants were analysed using metagenomic next-generation sequencing. Overall, seven different non-polio EV (NPEV) subtypes belonging to EV-B and EV-C species were identified, while viruses classified under EV-A and EV-D species could not be characterised at the sub-species level. Additionally, under EV-C species, two vaccine-related poliovirus subtypes (PV1 and PV3) were identified. The most prevalent NPEV species was EV-B (16/17, 94.1%), followed by EV-A (3/17, 17.6%), and EV-D (4/17, 23.5%). Within EV-B, the commonly identified NPEV types included echoviruses 6, 13, 15, and 19 (E6, E13, E15, and E19), and coxsackievirus B2 (CVB2), whereas enterovirus C99 (EV-C99) and coxsackievirus A19 (CVA19) were the only two NPEVs identified under EV-C species. Sabin PV1 and PV3 strains were predominantly detected during the first week of birth and 6-8 week time points, respectively, corresponding with the OPV vaccination schedule in South Africa. A total of 11 complete/near-complete genomes were identified from seven NPEV subtypes, and phylogenetic analysis of the three EV-C99 identified revealed that our strains were closely related to other strains from Cameroon and Brazil, suggesting global distribution of these strains. This study provides an insight into the frequency and diversity of EVs circulating in asymptomatic infants from the Free State Province, with the predominance of subtypes from EV-B and EV-C species. This data will be helpful to researchers looking into strategies for the control and treatment of EV infection.

Exploring the Pathogen Diagnosis and Prognostic Factors of Severe COVID-19 Using Metagenomic Next-Generation Sequencing: A Retrospective Study

Background: This study aimed to identify pathogens and factors that predict the outcome of severe COVID-19 by utilizing metagenomic next-generation sequencing (mNGS) technology. Methods: We retrospectively analyzed data from 56 severe COVID-19 patients admitted to our hospital between December 2022 and March 2023. We analyzed the pathogen types and strains detected through mNGS and conventional microbiological testing and collected general patient information. Results: In this study, 42 pathogens were detected using mNGS and conventional microbiological testing. mNGS had a significantly higher detection rate of 90.48% compared to 71.43% for conventional testing (P=0.026). A total of 196 strains were detected using both methods, with a significantly higher detection rate of 70.92% for mNGS compared to 49.49% for conventional testing (P=0.000). The 56 patients were divided into a survival group (33 cases) and a death group (23 cases) based on clinical outcomes. The survival group had significantly lower age, number of pathogens detected by mNGS, number of pathogens detected by conventional testing, APACHE-II score, SOFA score, high-sensitivity troponin, creatine kinase-MB subtype, and lactate dehydrogenase compared to the death group (P&lt;0.05). Multivariate logistic regression analysis showed that these factors were risk factors for mortality in severe COVID-19 patients (P&lt;0.05). In contrast, ROC curve analysis revealed that these factors had diagnostic values for mortality, with AUC values ranging from 0.657 to 0.963. The combined diagnosis of these indicators had an AUC of 0.924. Conclusion: The use of mNGS technology can significantly enhance the detection of pathogens in severe cases of COVID-19 and also has a solid ability to predict clinical outcomes.

Clinical application of nanopore-targeted sequencing technology in bronchoalveolar lavage fluid from patients with pulmonary infections.

The rapid and effective identification of pathogens in patients with pulmonary infections has posed a persistent challenge in medicine, with conventional microbiological tests (CMTs) proving time-consuming and less sensitive, hindering early diagnosis of respiratory infections. While there has been some research on the clinical performance of targeted sequencing technologies, limited focus has been directed toward bronchoalveolar lavage fluid (BALF). This study primarily evaluates the pathogen detection capabilities of nanopore-targeted sequencing (NTS) in BALF, providing a comprehensive analysis. The retrospective study, spanning from January 2022 to November 2023, includes 223 patients exclusively sourced from a single center. We conducted a detailed comparative analysis among NTS, targeted next-generation sequencing (tNGS), and CMTs. Initially, we compared the detection capabilities of NTS and tNGS and found no significant differences in their sensitivity and specificity. Specifically, we observed that the sensitivity of NTS was significantly higher than that of CMTs (74.83% vs 33.11%, P < 0.001). Furthermore, NTS exhibited a higher positivity rate in common pulmonary infections (62.88% vs. 23.48%) and in clinically suspected tuberculosis patients compared to CMTs (87.18% vs. 48.72%). Additionally, NTS showed less susceptibility to antibiotic interference, indicating a more sensitive detection capability, especially in detecting fastidious organisms. It complements GeneXpert in tuberculosis diagnosis and offers excellent advantages in identifying pathogens challenging for CMTs, such as non-tuberculous mycobacteria and viruses. Moreover, NTS significantly shortens the reporting time and is only a quarter of the cost of metagenomic next-generation sequencing. Clearly, NTS can facilitate faster and more cost-effective early diagnosis of respiratory infections.IMPORTANCEThis study holds paramount significance in advancing the field of respiratory infection diagnostics. By assessing the pathogen detection capabilities in bronchoalveolar lavage fluid (BALF) of patients with pulmonary infections, we illuminate the promising potential of nanopore-targeted sequencing (NTS). The findings underscore NTS as a comparable yet distinct alternative to traditional methods like comprehensive conventional microbiological tests (CMTs). Notably, NTS demonstrates a pivotal edge, expanding the spectrum of identified pathogens, particularly excelling in the detection of challenging entities like non-tuberculous mycobacteria and viruses. The study also highlights the complementary role of NTS alongside GeneXpert in the identification of tuberculosis, providing a comprehensive overview of the diagnostic landscape for respiratory infections. This insight carries significant implications for clinicians seeking rapid, cost-effective, and accurate diagnostic tools in the realm of pulmonary infections.

Application of Metagenomic Next-Generation Sequencing in the Diagnosis of Infectious Keratitis.

To determine the advantages of next-generation metagenomic sequencing (mNGS) technology in the diagnosis and treatment of infectious keratitis (IK). A total of 287 patients with IK admitted to the Department of Ophthalmology of Nanjing First Hospital between August 2018 and December 2022 were analyzed retrospectively, and the pathogenic causes, etiological characteristics, detection, treatment methods, and efficacy were summarized. Trauma and foreign matter were the most common causes of IK (144 patients, 50.2%). Of the 287 patients, 228 (79.4%) were diagnosed with a specific etiology, including 110 (48.2%) fungal infections, 44 (19.3%) viral infections, 42 (18.4%) mixed infections, and 30 (13.2%) bacterial infections. Filamentous fungi represented by Fusarium and Aspergillus were the most common, followed by bacteria such as Pseudomonas aeruginosa, Streptococcus pneumoniae, viruses (Herpes Simplex Virus/Varicella-Zoster Virus), and parasites. The positivity rates of secretion culture, corneal laser confocal microscopy (CM), mNGS, and pathological sections were 47.3% (133/281), 45.3% (111/245), 83.9% (104/124), and 19.3% (40/207), respectively. The positivity rate of mNGS for bacteria and viruses was higher than that of the other methods, and the positivity rate for fungi was the same as that for CM. As a result, 214 cases (74.6%) were cured, 51 cases (17.8%) improved, 8 cases (2.8%) did not heal, ocular content enucleation was performed in 14 cases (4.9%), and the overall efficacy rate was 92.3%. Trauma and foreign matter are the main causes of IK. The mNGS technology is an efficient and comprehensive detection method for viruses and bacteria, especially for mixed infections.

Current status and new experimental diagnostic methods of invasive fungal infections after hematopoietic stem cell transplantation.

Invasive fungal infections (IFIs) are common and life-threatening complications in post-hematopoietic stem cell transplantation (post-HSCT) recipients, Severe IFIs can lead to systemic infection and organ damage, which results in high mortality in HSCT recipients. With the development of the field of fungal infection diagnosis, more and more advanced non-culture diagnostic tools have been developed, such as glip biosensors, metagenomic next-generation sequencing, Magnetic Nanoparticles and Identified Using SERS via AgNPs+ , and artificial intelligence-assisted diagnosis. The advanced diagnostic approaches contribute to the success of HSCT and improve the overall survival of post-HSCT leukemia patients by supporting therapeutical decisions. This review provides an overview of the characteristics of two high-incidence IFIs in post-HSCT recipients and discusses some of the recently developed IFI detection technologies. Additionally, it explores the potential application of cationic conjugated polymer fluorescence resonance energy transfer (CCP-FRET) technology for IFI detection. The aim is to offer insights into selecting appropriate IFI detection methods and gaining an understanding of novel fungal diagnostic approaches in laboratory settings.

Application of metagenomic next-generation sequencing technology in the etiological diagnosis of peritoneal dialysis-associated peritonitis.

Pathogens detected by metagenomic next-generation sequencing (mNGS) and the laboratory blood culture flask method were compared to understand the advantages and clinical significance of mNGS assays in the etiological diagnosis of peritoneal dialysis-associated peritonitis (PDAP). The study involved a total of 37 patients from the hospital's peritoneal dialysis centre, six of whom were patients with non-peritoneal dialysis-associated peritonitis. Peritoneal dialysis samples were collected from the 37 patients, who were divided into two groups. One group's samples were cultured using conventional blood culture flasks, and the other samples underwent pathogen testing using mNGS. The results showed that the positive rate of mNGS was 96.77%, while that of the blood culture flask method was 70.97% (p < 0.05). A total of 29 pathogens were detected by mNGS, namely 24 bacteria, one fungus, and four viruses. A total of 10 pathogens were detected using the bacterial blood culture method, namely nine bacteria and one fungus. The final judgment of the PDAP's causative pathogenic microorganism was made by combining the clinical condition, response to therapy, and the whole-genome sequencing findings. For mNGS, the sensitivity was 96.77%, the specificity was 83.33%, the positive predictive value was 96.77%, and the negative predictive value was 83.33%. For the blood culture flask method, the sensitivity was 70.97%, the specificity was 100%, the positive predictive value was 100%, and the negative predictive value was 0%. In conclusion, mNGS had a shorter detection time for diagnosing peritoneal dialysis-related peritonitis pathogens, with a higher positive rate than traditional bacterial cultures, providing significant advantages in diagnosing rare pathogens.

Overlapping infections of Mycobacterium canariasense and Nocardia farcinica in an immunocompetent patient: A case report

BACKGROUND Infections by non-tuberculous mycobacteria (NTM) have become more common in recent years. Mycobacterium canariasense (M. canariasense ) was first reported as an opportunistic pathogen in 2004, but there have been very few case reports since then. Nocardia is a genus of aerobic and Gram-positive bacilli, and these species are also opportunistic pathogens and in the Mycobacteriales order. Conventional methods for diagnosis of NTM are inefficient. Metagenomic next-generation sequencing (mNGS) can rapidly detect many pathogenic microorganisms, even rare species. Most NTM and Nocardia infections occur in immunocompromised patients with atypical clinical symptoms. There are no previous reports of infection by M. canariasense and Nocardia farcinica (N. farcinica ), especially in immunocompetent patients. This case report describes an immunocompetent 52-year-old woman who had overlapping infections of M. canariasense , N. farcinica , and Candida parapsilosis (C. parapsilosis ) based on mNGS. CASE SUMMARY A 52-year-old woman presented with a productive cough and chest pain for 2 wk, and recurrent episodes of moderate-grade fever for 1 wk. She received antibiotics for 1 wk at a local hospital, and experienced defervescence, but the productive cough and chest pain persisted. We collected samples of a lung lesion and alveolar lavage fluid for mNGS. The lung tissue was positive for M. canariasense , N. farcinica , and C. parapsilosis , and the alveolar lavage fluid was positive for M. canariasense . The diagnosis was pneumonia, and application of appropriate antibiotic therapy cured the patient. CONCLUSION Etiological diagnosis is critical for patients with infectious diseases. mNGS can identify rare and novel pathogens, and does not require a priori knowledge.

Application value of metagenomic next-generation sequencing in hematological patients with high-risk febrile neutropenia.

Metagenomic next-generation sequencing (mNGS) is a novel non-invasive and comprehensive technique for etiological diagnosis of infectious diseases. However, its practical significance has been seldom reported in the context of hematological patients with high-risk febrile neutropenia, a unique patient group characterized by neutropenia and compromised immune responses. This retrospective study evaluated the results of plasma cfDNA sequencing in 164 hematological patients with high-risk febrile neutropenia. We assessed the diagnostic efficacy and clinical impact of mNGS, comparing it with conventional microbiological tests. mNGS identified 68 different pathogens in 111 patients, whereas conventional methods detected only 17 pathogen types in 36 patients. mNGS exhibited a significantly higher positive detection rate than conventional methods (67.7% vs. 22.0%, P < 0.001). This improvement was consistent across bacterial (30.5% vs. 9.1%), fungal (19.5% vs. 4.3%), and viral (37.2% vs. 9.1%) infections (P < 0.001 for all comparisons). The anti-infective treatment strategies were adjusted for 51.2% (84/164) of the patients based on the mNGS results. mNGS of plasma cfDNA offers substantial promise for the early detection of pathogens and the timely optimization of anti-infective therapies in hematological patients with high-risk febrile neutropenia.

Diagnosis model of early Pneumocystis jirovecii pneumonia based on convolutional neural network: a comparison with traditional PCR diagnostic method

BackgroundPneumocystis jirovecii pneumonia (PJP) is an interstitial pneumonia caused by pneumocystis jirovecii (PJ). The diagnosis of PJP primarily relies on the detection of the pathogen from lower respiratory tract specimens. However, it faces challenges such as difficulty in obtaining specimens and low detection rates. In the clinical diagnosis process, it is necessary to combine clinical symptoms, serological test results, chest Computed tomography (CT) images, molecular biology techniques, and metagenomics next-generation sequencing (mNGS) for comprehensive analysis.PurposeThis study aims to overcome the limitations of traditional PJP diagnosis methods and develop a non-invasive, efficient, and accurate diagnostic approach for PJP. By using this method, patients can receive early diagnosis and treatment, effectively improving their prognosis.MethodsWe constructed an intelligent diagnostic model for PJP based on the different Convolutional Neural Networks. Firstly, we used the Convolutional Neural Network to extract CT image features from patients. Then, we fused the CT image features with clinical information features using a feature fusion function. Finally, the fused features were input into the classification network to obtain the patient's diagnosis result.ResultsIn this study, for the diagnosis of PJP, the accuracy of the traditional PCR diagnostic method is 77.58%, while the mean accuracy of the optimal diagnostic model based on convolutional neural networks is 88.90%.ConclusionThe accuracy of the diagnostic method proposed in this paper is 11.32% higher than that of the traditional PCR diagnostic method. The method proposed in this paper is an efficient, accurate, and non-invasive early diagnosis approach for PJP.

Molecular rapid diagnostic testing for bloodstream infections: Nanopore targeted sequencing with pathogen-specific primers

Nanopore sequencing, known for real-time analysis, shows promise for rapid clinical infection diagnosis but lacks effective assays for bloodstream infections (BSIs). We prospectively assessed the performance of a novel nanopore targeted sequencing (NTS) assay in identifying pathogens and predicting antibiotic resistance in BSIs, analyzing 387 blood samples from December 2021 to April 2023. The positivity rate for NTS (69.5%, 269/387) nearly matches that of metagenomic next-generation sequencing (mNGS) (74.7%, 289/387; p=0.128) and surpasses the positivity rate of conventional blood culture (BC) (33.9%, 131/387; p<0.01). Frequent pathogens detected by NTS included Klebsiella pneumoniae (n=54), Pseudomonas aeruginosa (n=36), Escherichia coli (n=36), Enterococcus faecium(n=30), Acinetobacter baumannii(n=26), Staphylococcus aureus(n=23), and Human cytomegalovirus (n=37). Against a composite BSI diagnostic standard, NTS demonstrated a sensitivity and specificity of 84.0% (95% CI 79.5%-87.7%) and 90.1% (95% CI 81.7%-88.5%), respectively. The concordance between NTS and mNGS results (the percentage of total cases where both either detected BSI-related pathogens or were both negative) was 90.2% (359/387), whereas the consistency between NTS and BC was only 60.2% (233/387). In 80.6% (50/62) of the samples with identical pathogens identified by both NTS tests and BCs, the genotypic resistance identified by NTS correlated with culture-confirmed phenotypic resistance. Using NTS, 95% of samples can be tested and analyzed in approximately 7h, allowing for early patient diagnosis. NTS is rapid, sensitive, and efficient for detecting BSIs and drug-resistant genes, making it a potential preferred diagnostic tool for early infection identification in critically ill patients.

The clinical value of mNGS of bronchoalveolar lavage fluid versus traditional microbiological tests for pathogen identification and prognosis of severe pneumonia (NT-BALF):study protocol for a prospective multi-center randomized clinical trial.

Early, rapid, and accurate pathogen diagnosis can help clinicians select targeted treatment options, thus improving prognosis and reducing mortality rates of severe pneumonia. Metagenomic next-generation sequencing (mNGS) has a higher sensitivity and broader pathogen spectrum than traditional microbiological tests. However, the effects of mNGS-based antimicrobial treatment procedures on clinical outcomes and cost-effectiveness in patients with severe pneumonia have not been evaluated. This is a regional, multi-center, open, prospective, randomized controlled trial to evaluate that whether the combination of mNGS and traditional testing methods could decrease 28-day call-cause mortality with moderate cost-effectiveness. A total of 192 patients with severe pneumonia will be recruited from four large tertiary hospitals in China. Bronchoalveolar lavage fluid will be obtained in all patients and randomly assigned to the study group (mNGS combined with traditional microbiological tests) or the control group (traditional microbiological tests only) in a 1:1 ratio. Individualized antimicrobial treatment and strategy will be selected according to the analysis results. The primary outcome is 28-day all-cause mortality. The secondary outcomes are ICU and hospital length of stay (LOS), ventilator-free days and ICU-free days, consistency between mNGS and traditional microbiological tests, detective rate of mNGS and traditional microbiological tests, turn-out time, time from group allocation to start of treatment, duration of vasopressor support, types and duration of anti-infective regimens, source of drug-resistant bacteria or fungi, and ICU cost. The clinical benefits of mNGS are potentially significant, but its limitations should also be considered. ChineseClinicalTrialRegistry.org, ChiCTR2300076853. Registered on 22 October 2023.

Application of metagenomic next-generation sequencing in the diagnosis of Bartonella neuroretinitis: a case report and literature review.

Cat-scratch disease (CSD) is caused by Bartonella henselae infection. In atypical cases of CSD, pathogen determination is challenging. We report a case of Bartonella neuroretinitis with neither a clear history of scratches nor typical general symptoms. The diagnosis was made using metagenomic next-generation sequencing (mNGS), a high-throughput sequencing technology. A female patient presented to the ophthalmologist with complaint of blurred vision in her right eye. Although with history of raising a cat, she reported no clear history of scratches or typical general symptoms, except a fever of unknown origin which resolved spontaneously. The best corrected visual acuity (BCVA) of the right eye was count fingers. Fundus examination showed optic disc oedema, macular exudates and inferior exudative retinal detachment. Laboratory examination results showed increased value of serum C-reactive protein and erythrocyte sedimentation rate. Ocular involvement of toxoplasmosis, syphilis and tuberculosis were excluded. To identify the possible causative pathogen of the disease, mNGS of aqueous humour sample was performed and 521 reads of B. henselae were identified. Serological test results further showed a positive immunoglobulin G (IgG) titre of 1:64. Taking the contact history, clinical manifestations, mNGS and serological results into consideration, the diagnosis of Bartonella neuroretinitis (ocular CSD) was made. After appropriatetreatment, the BCVA of the right eye improved to 20/25 in the last follow-up. Fundus examination showed a normal optic disc and macula, and the exudates had reduced. mNGS, a fast and unbiased method, can be used to detect B. henselae (if present) in intraocular fluid samples.; however, the results should be interpreted together with the clinical symptoms and other auxiliary test results.

Clinical application of metagenomic next-generation sequencing for the diagnosis of suspected infection in adults: A cross-sectional study.

Metagenomic next-generation sequencing (mNGS) has become an available method for pathogen detection. The clinical application of mNGS requires further evaluation. We conducted a cross-sectional study of 104 patients with suspected infection between May 2019 and May 2021. The risk factors associated with infection were analyzed using univariate logistic analysis. The diagnostic performance of pathogens was compared between mNGS and conventional microbiological tests. About 104 patients were assigned into 3 groups: infected group (n = 69), noninfected group (n = 20), and unknown group (n = 15). With the composite reference standard (combined results of all microbiological tests, radiological testing results, and a summary of the hospital stay of the patient) as the gold standard, the sensitivity, specificity, positive predictive value, negative predictive value of mNGS was 84.9%, 50.0%, 88.6%, and 42.1%, respectively. Compared with conventional microbiological tests, mNGS could detect more pathogens and had obvious advantages in Mycobacterium tuberculosis, Aspergillus, and virus detection. Moreover, mNGS had distinct benefits in detecting mixed infections. Bacteria-fungi-virus mixed infections were the most common in patients with severe pneumonia. mNGS had a higher sensitivity than conventional microbiological tests, especially for M. tuberculosis, Aspergillus, viruses, and mixed infections. We suggest that mNGS should be used more frequently in the early diagnosis of pathogens in critically ill patients in the future.

Unraveling the potential of metagenomic next-generation sequencing in infectious disease diagnosis: Challenges and prospects

Unraveling the potential of metagenomic next-generation sequencing in infectious disease diagnosis: Challenges and prospects

The complete genome sequence of unculturable Mycoplasma faucium obtained through clinical metagenomic next-generation sequencing.

Diagnosing Mycoplasma faucium poses challenges, and it's unclear if its rare isolation is due to infrequent occurrence or its fastidious nutritional requirements. This study analyzes the complete genome sequence of M. faucium, obtained directly from the pus of a sternum infection in a lung transplant patient using metagenomic sequencing. Genome analysis revealed limited therapeutic options for the M. faucium infection, primarily susceptibility to tetracyclines. Three classes of mobile genetic elements were identified: two new insertion sequences, a new prophage (phiUMCG-1), and a species-specific variant of a mycoplasma integrative and conjugative element (MICE). Additionally, a Type I Restriction-Modification system was identified, featuring 5'-terminally truncated hsdS pseudogenes with overlapping repeats, indicating the potential for forming alternative hsdS variants through recombination. This study represents the first-ever acquisition of a complete circularized bacterial genome directly from a patient sample obtained from invasive infection of a primary sterile site using culture-independent, PCR-free clinical metagenomics.

Antimicrobial resistance prediction by clinical metagenomics in pediatric severe pneumonia patients

BackgroundAntimicrobial resistance (AMR) is a major threat to children’s health, particularly in respiratory infections. Accurate identification of pathogens and AMR is crucial for targeted antibiotic treatment. Metagenomic next-generation sequencing (mNGS) shows promise in directly detecting microorganisms and resistance genes in clinical samples. However, the accuracy of AMR prediction through mNGS testing needs further investigation for practical clinical decision-making.MethodsWe aimed to evaluate the performance of mNGS in predicting AMR for severe pneumonia in pediatric patients. We conducted a retrospective analysis at a tertiary hospital from May 2022 to May 2023. Simultaneous mNGS and culture were performed on bronchoalveolar lavage fluid samples obtained from pediatric patients with severe pneumonia. By comparing the results of mNGS detection of microorganisms and antibiotic resistance genes with those of culture, sensitivity, specificity, positive predictive value, and negative predictive value were calculated.ResultsmNGS detected bacterial in 71.7% cases (86/120), significantly higher than culture (58/120, 48.3%). Compared to culture, mNGS demonstrated a sensitivity of 96.6% and a specificity of 51.6% in detecting pathogenic microorganisms. Phenotypic susceptibility testing (PST) of 19 antibiotics revealed significant variations in antibiotics resistance rates among different bacteria. Sensitivity prediction of mNGS for carbapenem resistance was higher than penicillins and cephalosporin (67.74% vs. 28.57%, 46.15%), while specificity showed no significant difference (85.71%, 75.00%, 75.00%). mNGS also showed a high sensitivity of 94.74% in predicting carbapenem resistance in Acinetobacter baumannii.ConclusionsmNGS exhibits variable predictive performance among different pathogens and antibiotics, indicating its potential as a supplementary tool to conventional PST. However, mNGS currently cannot replace conventional PST.

The clinical importance of metagenomic next-generation sequencing in detecting disease-causing microorganisms in cases of sepsis acquired in the community or hospital setting.

Although metagenomic next-generation sequencing (mNGS) is commonly used for diagnosing infectious diseases, clinicians face limited options due to the high costs that are not covered by basic medical insurance. The goal of this research is to challenge this bias through a thorough examination and evaluation of the clinical importance of mNGS in precisely identifying pathogenic microorganisms in cases of sepsis acquired in the community or in hospitals. A retrospective observational study took place at a tertiary teaching hospital in China from January to December 2021. Data on 308 sepsis patients were collected, and the performance of etiological examination was compared between mNGS and traditional culture method. Two hundred twenty-nine cases were observed in the community-acquired sepsis (CAS) group and 79 cases in the hospital-acquired sepsis (HAS) group. In comparison with conventional culture, mNGS showed a significantly higher rate of positivity in both the CAS group (88.21% vs. 25.76%, adj.P < 0.001) and the HAS group (87.34% vs. 44.30%, adj.P < 0.001), particularly across various infection sites and specimens, which were not influenced by factors like antibiotic exposure or the timing and frequency of mNGS technology. Sepsis pathogens detected by mNGS were broad, especially viruses, Mycobacterium tuberculosis, and atypical pathogens, with mixed pathogens being common, particularly bacterial-viral co-detection. Based on the optimization of antimicrobial therapy using mNGS, 58 patients underwent antibiotic de-escalation, two patients were switched to antiviral therapy, and 14 patients initiated treatment for tuberculosis, resulting in a reduction in antibiotic overuse but without significant impact on sepsis prognosis. The HAS group exhibited a critical condition, poor prognosis, high medical expenses, and variations in etiology, yet the mNGS results did not result in increased medical costs for either group. mNGS demonstrates efficacy in identifying multiple pathogens responsible for sepsis, with mixed pathogens of bacteria and viruses being prevalent. Variability in microbiological profiles among different infection setting underscores the importance of clinical vigilance. Therefore, the adoption of mNGS for microbiological diagnosis of sepsis warrants acknowledgment and promotion.