Utility Of Metagenomic Next-generation Sequencing Research Articles

Pathogen spectrum and microbiome in lower respiratory tract of patients with different pulmonary diseases based on metagenomic next-generation sequencing.

The homeostasis of the microbiome in lower respiratory tract is crucial in sustaining normal physiological functions of the lung. Different pulmonary diseases display varying degrees of microbiome imbalance; however, the specific variability and clinical significance of their microbiomes remain largely unexplored. In this study, we delineated the pathogen spectrum and commensal microorganisms in the lower respiratory tract of various pulmonary diseases using metagenomic sequencing. We analyzed the disparities and commonalities of the microbial features and examined their correlation with disease characteristics. We observed distinct pathogen profiles and a diversity in lower airway microbiome in patients diagnosed with cancer, interstitial lung disease, bronchiectasis, common pneumonia, Nontuberculous mycobacteria (NTM) pneumonia, and severe pneumonia. This study illustrates the utility of Metagenomic Next-generation Sequencing (mNGS) in identifying pathogens and analyzing the lower respiratory microbiome, which is important for understanding the microbiological aspect of pulmonary diseases and essential for their early and precise diagnosis.

Clinical Utility of Metagenomic Next-Generation Sequencing of Plasma Microbial Cell-Free DNA: a Single-Center Retrospective Study

Abstract Infectious diseases account for significant morbidity and mortality in healthcare, and metagenomic next-generation sequencing of plasma microbial cell-free DNA has emerged as a promising non-invasive diagnostic tool in this scenario. However, the available data on the real impact of this test in clinical practice is limited and conflicting. In this context, we performed a retrospective evaluation of the utilization of this test at our institution since February 17, 2019. A total of 76 tests were performed in this period in 68 different patients. The mean age of the patients was 9 years old, with 40 being male (58.82%) and 28 female (41.18%). The ordering specialties included infectious diseases (82.90%), hematology/oncology (9.21%), neurology (3.95%), pulmonology (2.63%), and dermatology (1.31%). The vast majority of the tests were ordered as inpatient (73 tests, 96.05%). 45 tests (59.21%) resulted as positive, of which 19 (42.22%) were polymicrobial. The tests were concordant with conventional microbiological studies 47 times (61.84%). Most tests (67 tests, 88.16%) had no significant clinical impact, 8 tests (10.53%) had a positive impact, and one test (1.31%) had an indeterminate impact. No tests were determined to have a negative clinical impact. Overall, our data is consistent with most studies which assessed the clinical utility of metagenomic next-generation sequencing of plasma microbial cell-free DNA in the literature, showing that testing did not have a significant impact on patient care for the majority of cases. There were some cases, however, in which testing showed a positive impact in escalating or de-escalating antibiotic therapy. In conclusion, despite being mostly a bystander, in selected cases and in the right clinical context, the test can be useful to guide antimicrobial treatment without causing significant harm. More studies are needed to further investigate the real utility of the test.

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.

Clinical utility of metagenomic next-generation sequencing on bronchoalveolar lavage fluid in diagnosis of lower respiratory tract infections

BackgroundIn this study, we aimed to evaluate the clinical utility of Metagenomic Next-Generation sequencing (mNGS) on bronchoalveolar lavage fluid (BALF) in diagnosis of Lower Respiratory Tract Infections (LRTIs).MethodsIn this study, we retrospectively analyzed 186 hospitalized patients who were suspected with LRTIs and performed mNGS (DNA) test of BALF simultaneously at The Fifth Affiliated Hospital of Sun Yat-Sen University from March 2023 to August 2023. Suspected LRTI was based on LRTI related clinical manifestations or imaging examination. Among them, 155 patients had undergone conventional culture and mNGS (DNA) testing simultaneously. Finally, 138 cases (89.03%,138/155) were diagnosed as LRTI and 17 cases (10.97%,17/155) were diagnosed as non-LRTI. Both detecting rate and diagnostic efficacy of mNGS and conventional culture were compared.ResultsThe positive detection rates of pathogens between mNGS and conventional culture were significant different (81.29% VS 39.35%, P < 0.05). Compared with paired conventional culture result, the sensitivity of mNGS in diagnosis of LRTIs was more superior (88.41% VS 43.48%; P < 0.05), the specificity was opposite (76.47% VS 94.12%; P > 0.05). Furthermore, 77.54% and 35.51% of LRTI cases were being etiologically diagnosed by mNGS and culture respectively. Importantly, mNGS directly led to a change of treatment regimen in 58 (37.42%) cases, including antibiotic adjustment (29.68%) and ruling out active infection (7.74%). Moreover, treatment regimen remained unchanged in 97 (62.58%) cases, considering the current antibiotic therapy already covered the detected pathogens (36.13%) or empirical treatment was effective (11.61%).ConclusionsmNGS can identify a wide range of pathogens in LRTIs, with improved sensitivity and being more superior at diagnosing LRTIs etiologically. mNGS has the potential to enhance clinical outcomes by optimizing the treatment regimens.

Clinical utility of metagenomic next-generation sequencing in fever of undetermined origin.

Metagenomic next-generation sequencing (mNGS) is a novel diagnostic tool increasingly used in the field of infectious diseases. Little guidance is available regarding its appropriate use in different patient populations and clinical syndromes. We aimed to review the clinical utility of mNGS in patients with a specific clinical syndrome and identify factors that may increase its utility. We retrospectively reviewed charts of 72 non-immunocompromised adults hospitalized with the clinical syndrome of 'fever of undetermined origin' and underwent mNGS testing. Standardized criteria from a previously published study were used to determine the clinical impact of mNGS testing. We applied logistic regression to identify factors associated with a positive clinical impact. Of the 72 patients identified, 62.5% were males with a median age of 56. All patients had a fever at the time of evaluation. At least one organism was identified in 65.3% of cases; most commonly were Epstein-Barr virus (13.9%), cytomegalovirus (12.5%), and Rickettsia typhi (11.1%). Of those determined to have an infectious etiology of their febrile syndrome, 89.5% (n = 34/38) had a positive mNGS. Consistency between the organism(s) on mNGS and the clinically determined infectious etiology was 82.4%. mNGS had a positive clinical impact in 40.3% of cases, a negative impact in 2.8%, and no impact in 56.9% of cases. Besides age, we did not identify other factors associated with a higher likelihood of positive clinical impact. In our review, mNGS had a positive clinical impact in a large proportion of adults with fever of undetermined origin, with minimal negative impact. However, mNGS results should be interpreted carefully given the high rate of detection of pathogens of unclear clinical significance. Randomized clinical trials are needed to assess the clinical utility of this novel diagnostic tool.

Clinical utility of metagenomic next-generation sequencing in the diagnosis of invasive pulmonary aspergillosis in acute exacerbation of chronic obstructive pulmonary disease patients in the intensive care unit.

To explore the clinical utility of metagenomic next-generation sequencing (mNGS) in diagnosing invasive pulmonary aspergillosis (IPA) among patients with acute exacerbation of chronic obstructive pulmonary disease (AECOPD) in the intensive care unit (ICU). A retrospective analysis was conducted on patients with AECOPD admitted to the ICU of Xinxiang Central Hospital in Henan Province, China, between March 2020 and September 2023, suspected of having IPA. Bronchoalveolar lavage fluid (BALF) samples were collected for fungal culture, the galactomannan (GM) test, and mNGS. Based on host factors, clinical features, and microbiological test results, patients were categorized into 62 cases of IPA and 64 cases of non-IPA. Statistical analysis was performed to compare the diagnostic efficacy of fungal culture, the serum and BALF GM test, and mNGS detection for IPA in patients with AECOPD. 1. The sensitivity and specificity of mNGS in diagnosing IPA were 70.9% and 71.8% respectively, with the sensitivity of mNGS surpassing that of fungal culture (29.0%, P<0.01), serum GM test (35.4%, P<0.01), and BALF GM test (41.9%, P<0.05), albeit with slightly lower specificity compared to fungal culture (90.6%, P >0.05), serum GM test (87.5%, P >0.05), and BALF GM test (85.9%, P >0.05).Combining fungal culture with the GM test and mNGS resulted in a sensitivity of 80.6% and a specificity of 92.2%, underscoring a superior diagnostic rate compared to any single detection method. 2.mNGS accurately distinguished strains of the Aspergillus genus. 3.The area under the ROC curves of mNGS was 0.73, indicating good diagnostic performance. 4.The detection duration for mNGS is shorter than that of traditional fungal culture and GM testing. mNGS presents a pragmatic and highly sensitive approach, serving as a valuable complementary tool to conventional microbiological tests (CMT). Our research demonstrated that, compared to fungal culture and GM testing, mNGS exhibits superior diagnostic capability for IPA among patients with AECOPD. Integration of mNGS with established conventional methods holds promise for improving the diagnosis rate of IPA.

Utility of Metagenomic Next-Generation Sequencing for Diagnosis of Infectious Diseases in Critically Ill Immunocompromised Pediatric Patients.

Infections cause high rates of illness and death in children worldwide. However, studies on the clinical value of metagenomic next-generation sequencing (mNGS) for immunocompromised children are still limited. From June 2021 to December 2023, 119 samples were collected at Pediatric Intensive Care Unit (PICU) of a single-center pediatric hospital and classified into two groups based on their immune states. We compared the diagnostic performance of mNGS and conventional microbiological test (CMT) for pathogen identification, and assessed the clinical impacts of mNGS. Among the 119 samples, 48 (40.34%) belonged to the immunocompromised children. mNGS had a higher positivity rate than CMT (76.47% vs 55.46%, P = 0.0006). The positive percent agreement (PPA) of mNGS for immunocompromised children was higher compared to immunocompetent children (95.24% vs 77.78%). The most common pathogens for immunocompromised patients were gram-negative bacteria and herpesvirus. However, immunocompetent children showed a higher detection rate for gram-positive bacteria and respiratory viruses. Furthermore, the proportions of the positive impact of mNGS results were significantly higher in immunocompromised patients compared to immunocompetent patients for both diagnosis (91.67% vs 57.75%) and treatment (95.83% vs 64.79%) (P < 0.0001). Immunocompromised state, length of hospital stays, times stay in ICU, Pediatric Risk of Mortality (PRISM) score, neutrophil percentage (NEUT%) and the ratio of arterial oxygen partial pressure to fractional inspired oxygen (PaO2/FiO2) were considered independent factors for poor prognosis in critically ill pediatric patients. In patients from PICU, mNGS had a greater clinical significance in immunocompromised children compared to immunocompetent children. mNGS technology is an important auxiliary method for achieving accurate diagnosis and treatment of critically ill pediatric patients.

Clinical utility of metagenomic next-generation sequencing in pathogen detection for lower respiratory tract infections and impact on clinical outcomes in southernmost China.

Today, metagenomic next-generation sequencing (mNGS) has emerged as a diagnostic tool for infections. However, since Hainan has a complicated pathogen spectrum, the diagnostic value and impact on patient outcomes of mNGS in Hainan are to be explored. From April 2020 to October 2021, 266 suspected lower respiratory tract infections (LRTIs) patients in Hainan were enrolled, and specimens were collected before antibiotic treatment. Bronchoalveolar lavage fluid (BALF) samples were subjected to mNGS and culture to compare the diagnostic performance. Other conventional microbiological tests (CMT) were also performed. Patients' treatments and clinical outcomes were recorded, and the antibiotic resistance genes (ARGs) were detected via mNGS workflow. The positive rate of mNGS outperformed that of culture (87.55% vs. 39.30%, p<0.001) and CMT (87.12% vs. 52.65%, p<0.001). Specifically, mNGS detected more P. aeruginosa (12.03% vs 9.02%, p<0.05), H. influenzae (9.77% vs 2.26%, p<0.001), Aspergillus fumigatus (3.00% vs 0.75%, p<0.05), Candida albicans (26.32% vs 7.52%, p<0.001) and uncommon pathogens. It also demonstrated great diagnostic advantages in Mycobacterium tuberculosis with 80% sensitivity and 97.4% specificity. Over half of the patients (147, 55.26%) had modified empirical treatment according to mNGS results and 89.12% of them responded well. For three deaths with modified treatment, multiple drug resistance was predicted by mNGS and confirmed by antibiotic susceptibility test. The application of mNGS can benefit clinics in pathogen identification and antimicrobial treatment stewardship. Physicians should be alert to some emerging uncommon pathogens, including Chlamydia Psittaci, Nocardia otitidiscaviarum, and rare NTM.

A systematic review and meta-analysis of the diagnostic accuracy of metagenomic next-generation sequencing for diagnosing tuberculous meningitis.

The utility of metagenomic next-generation sequencing (mNGS) in the diagnosis of tuberculous meningitis (TBM) remains uncertain. We performed a meta-analysis to comprehensively evaluate its diagnostic accuracy for the early diagnosis of TBM. English (PubMed, Medline, Web of Science, Cochrane Library, and Embase) and Chinese (CNKI, Wanfang, and CBM) databases were searched for relevant studies assessing the diagnostic accuracy of mNGS for TBM. Review Manager was used to evaluate the quality of the included studies, and Stata was used to perform the statistical analysis. Of 495 relevant articles retrieved, eight studies involving 693 participants (348 with and 345 without TBM) met the inclusion criteria and were included in the meta-analysis. The pooled sensitivity, specificity, positive likelihood ratio, negative likelihood ratio, diagnostic odds ratio, and area under the summary receiver-operating characteristic curve of mNGS for diagnosing TBM were 62% (95% confidence interval [CI]: 0.46-0.76), 99% (95% CI: 0.94-1.00), 139.08 (95% CI: 8.54-2266), 0.38 (95% CI: 0.25-0.58), 364.89 (95% CI: 18.39-7239), and 0.97 (95% CI: 0.95-0.98), respectively. mNGS showed good specificity but moderate sensitivity; therefore, a more sensitive test should be developed to assist in the diagnosis of TBM.

The clinical utility of metagenomic next-generation sequencing for the diagnosis of central nervous system infectious diseases.

To evaluate the clinical utility of metagenomic next-generation sequencing (mNGS) for the diagnosis of central nervous system infections (CNSI). Cerebrospinal fluid (CSF) from 54 patients who were high-level clinical suspicion of CNSI was collected and sent for mNGS and conventional tests from January 2019 to March 2022. Twenty out of 54 patients were diagnosed with CNSI and 34 non-CNSI. Among the 34 non-CNSI, one was false positive by mNGS. Among the 20 CNSI, 11 had presumed viral encephalitis and/or meningitis, 5 had presumed bacterial meningitis, 2 had presumed TMB, 1 had Crytococcus meningitis and 1 had neurosyphilis. The sensitivity of viral encephalitis and/or meningitis was 0.73 (8/11); 10 virus were detected; 9/10 was dsDNA; 1/10 was ssRNA. SSRN ranged from 1 to 13. The accuracy rate was 0.4, the accuracy rate was positively correlated with SSRN (r = 0.738, P = 0.015), SSRN ≥ 1, the accuracy rate was 0.4; SSRN ≥ 3, the accuracy rate was 0.66; SSRN ≥ 4, the accuracy rate was 0.75; SSRN ≥ 6, the accuracy rate was 1. The sensitivity of bacterial meningitis was 1. Seven kinds of bacteria were detected, among which 3/7 were gram positive, 3/7 were gram negative, and 1/7 was infected NTM (nontuberculous mycobacteria). The accuracy rate was 0.43 (3/7). The sensitivity of TBM was 0.66 (2/3), the accuracy rate was 1. The sensitivity of Crytococcus meningitis was 1, the accuracy rate was 0.5. PPV (positive predictive value) of mNGS was 0.94, NPV (negative predictive value) of mNGS was 0.89, specificity was 0.97 and sensitivity was 0.8. The AUG for CSF mNGS diagnosis of CNSI was 0.89 (95% CI = 0.78-0.99) Headache, meningeal irritation sign and image of meninges abnormal were correlated with the sensitivity of mNGS (r = 0.451, 0.313, 0.446; p = 0.001, 0.021, 0.001); CSF Glucose and CSF Chloride were negatively correlated with sensitivity of mNGS (r = -0.395, -0.462; p = 0.003, ＜ 0.001). mNGS is a detection means with high sensitivity, wide coverage and strong timeliness, which can help clinicians to identify the pathogen diagnosis quickly, conduct targeted anti-infection treatment early and reduce antibiotic abuse. The pathogen which causing low CSF Glucose, low CSF Chloride or meninges infections was more likely to be detected by mNGS. It may be related to growth and structural characteristics of the pathogen and blood-brain barrier damage.

The Utility of Metagenomic Next-Generation Sequencing (mNGS) in the Management of Patients With Bronchiectasis: A Single-Center Retrospective Study of 93 Cases.

Bronchiectasis is a chronic inflammatory respiratory disease mainly caused by pathogenic infections. However, standard methods of pathogen detection show prolonged cycle durations and unsatisfactory sensitivity and detection rates. Macrogenomic next-generation sequencing (mNGS) emerges as a promising technique for swift, effective, and unbiased pathogen detection and subsequent data interpretation. Here, a retrospective analysis of 93 patients with suspected bronchiectasis was performed to assess the clinical applicability of mNGS. Bronchoalveolar alveolar lavage fluid (BALF) samples were collected from these subjects, followed by standard assays and mNGS separately. The turnaround time, detection rate, and pathogen identification using mNGS were compared with those of standard methods. mNGS identified a greater number of bacteria (72 vs 16), fungi (26 vs 19), and viruses (14 vs 0) than standard methods. Specifically, the commonly identified bacteria were Haemophilus, Mycobacterium intracellulare, Pseudomonas, and Streptococcus pneumoniae, while the most detected fungi were Aspergillus and the most prevalent viruses were human herpesviruses. Of note, 29 out of 30 patients (96.67%) who received optimized treatment strategies based on mNGS results experienced recovery. Collectively, these findings suggest that mNGS has the potential to improve the diagnosis and treatment of bronchiectasis patients by enabling rapid and precise pathogen detection, which can lead to timely and effective treatment strategies.

Application of metagenomic next-generation sequencing in the diagnosis and resistome analysis of community-acquired pneumonia pathogens from bronchoalveolar lavage samples.

To perform a prospective diagnostic study exploring the clinical utility of metagenomic next-generation sequencing (mNGS) in diagnosing community-acquired pneumonia (CAP), and revealing resistome differences in bronchoalveolar lavage fluid (BALF) from CAP patients with varying severity of admission base on Pneumonia Patient Outcomes Research Team (PORT) risk classes. We compared the diagnostic performances of mNGS and conventional testing for the detection of pathogens in BALF from 59 CAP patients, and performed resistome differences analysis of metagenomic data from 59 BALF samples, namely, 25 from CAP patients with PORT score I (I group), 14 from CAP patients with PORT score II (II group), 12 from CAP patients with PORT score III (III group), and 8 from CAP patients with PORT score IV (IV group). The diagnostic sensitivities of mNGS and conventional testing for the detection of pathogens in BALF in patients with CAP were 96.6% (57/59) and 30.5% (18/59), respectively. There was a significant difference in the overall relative abundance of resistance genes between the four groups (P=0.014). The results of principal coordinate analysis based on Bray-Curtis dissimilarities showed that there were significant differences in the composition of resistance genes among the I, II, III, and IV groups (P=0.007). A large number of antibiotic resistance genes, such as those affiliated with multidrug, tetracycline, aminoglycoside, and fosfomycin resistance, were enriched in the IV group. In conclusion, mNGS has a high diagnostic value in CAP. There were significant differences present in microbiota resistance to antibiotics in BALF from CAP patients in different PORT risk classes, which should attract enough attention.

Application of metagenomic next-generation sequencing in the diagnosis of urinary tract infection in patients undergoing cutaneous ureterostomy.

Urinary tract infection (UTI) is an inflammatory response of the urothelium to bacterial invasion and is a common complication in patients with cutaneous ureterostomy (CU). For such patients, accurate and efficient identification of pathogens remains a challenge. The aim of this study included exploring utility of metagenomic next-generation sequencing (mNGS) in assisting microbiological diagnosis of UTI among patients undergoing CU, identifying promising cytokine or microorganism biomarkers, revealing microbiome diversity change and compare virulence factors (VFs) and antibiotic resistance genes (ARGs) after infection. We performed a case-control study of 50 consecutive CU patients from December 2020 to January 2021. According to the clinical diagnostic criteria, samples were divided into infected group and uninfected group and difference of urine culture, cytokines, microorganism, ARGs and VFs were compared between the two groups. Inflammatory responses were more serious in infected group, as evidenced by a significant increase in IFN-α (p=0.031), IL-1β (0.023) and IL-6 (p=0.018). Clinical culture shows that there is higher positive rate in infected group for most clinical pathogens like Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus, Candida auris etc. and the top three pathogens with positive frequencies were E. coli, K. pneumoniae, and Enterococcus faecalis. Benchmarking clinical culture, the total sensitivity is 91.4% and specificity is 76.3% for mNGS. As for mNGS, there was no significant difference in microbiome α- diversity between infected and uninfected group. Three species biomarkers including Citrobacter freundii, Klebsiella oxytoca, and Enterobacter cloacae are enriched in infected group based on Lefse. E. cloacae were significantly correlated with IL-6 and IL-10. K. oxytoca were significantly correlated with IL-1β. Besides, the unweighted gene number and weighted gene abundance of VFs or ARGs are significantly higher in infected group. Notablely, ARGs belonging to fluoroquinolones, betalatmas, fosfomycin, phenicol, phenolic compound abundance is significantly higher in infected group which may have bad effect on clinical treatment for patients. mNGS, along with urine culture, will provide comprehensive and efficient reference for the diagnosis of UTI in patients with CU and allow us to monitor microbial changes in urine of these patients. Moreover, cytokines (IL-6, IL-1β, and IFN-a) or microorganisms like C. freundii, K. oxytoca or E. cloacae are promising biomarkers for building effective UTI diagnostic model of patients with CU and seriously the VFs and ARGs abundance increase in infected group may play bad effect on clinical treatment.

Human pegivirus identified in severe myelitis and optic neuritis in immunocompromised patients: A pathogenic role for a forgotten virus?

The role of Human pegivirus (HPgV) in patients with encephalitis has been recently questioned. We present cases of 4 patients with similar clinical, biological, and radiological characteristics, including a past history of transplantation with long-term immunosuppression and a progressive course of severe and predominantly myelitis, associated in 3 cases with optic neuropathy causing blindness. Extensive workup was negative but analysis of the CSF by use of pan-microorganism DNA- and RNA-based shotgun metagenomics was positive for HPgV. This case series further supports the hypothesis of HPgV CNS infection and highlights the utility of metagenomic next-generation sequencing of CSF in immunocompromised patients.

Utility of Metagenomic Next-Generation Sequencing for Etiological Diagnosis of Patients with Sepsis in Intensive Care Units.

ABSTRACTThe performance of metagenomic next-generation sequencing (mNGS) was evaluated and compared with that of conventional culture testing in patients with sepsis. Prospective blood and bronchoalveolar lavage fluid (BALF) samples from 50 patients with sepsis were tested using cultures (bacterial, fungal, and viral) and mNGS of microbial DNA (blood and BALF) and RNA (BALF). mNGS had higher detection rates than blood culture (88.0% versus 26.0%, P < 0.001) and BALF culture (92.0% versus 76.0%, P = 0.054). RNA-based mNGS has increased the detection rate of several bacteria, fungi, and viruses, but not mycobacteria and Toxoplasma gondii. The number of multiple detections per specimen was higher in BALF (92.0%) than in blood (78.0%) samples, and the highest number of pathogens detected in a single specimen was 32. Among blood samples, compared to cultures, mNGS detected significantly more bacteria (P < 0.001), fungi (P = 0.012), and viruses (P < 0.001), whereas BALF mNGS had a higher detection rate for bacteria (P < 0.001) and viruses (P < 0.001). The percentage of mNGS-positive samples was significantly higher than that of culture-positive samples for several Gram-negative bacteria, some Gram-positive bacteria, and viruses, but not fungi. Mycobacteria had a higher detection rate by culture than by mNGS, but the difference was not significant due to the small sample size. The positive and negative agreements with 95% confidence intervals of mNGS and culture were 62.0% (50.4 to 72.7) and 96.8% (96.5 to 97.1), respectively. mNGS offers a sensitive diagnostic method for patients with sepsis and is promising for the detection of multipathogen infections. Clinical correlation is advised to interpret mNGS data due to the lack of unified diagnostic criteria.IMPORTANCE Delays in effective antimicrobial therapy have resulted in decreased survival rates among patients with sepsis. However, current culture-based diagnostic methods have low sensitivity because of concurrent antibiotic exposure and fastidious and atypical causative organisms. Among patients with sepsis, we showed that mNGS methods had higher positive rates than culture methods, especially for bacteria, viruses, and multipathogen infections, which are difficult to culture and detect in patients treated with antibiotics. RNA-based mNGS has increased the detection rate of several bacteria, fungi, and viruses, but not mycobacteria and Toxoplasma gondii. mNGS also showed a high negative percent agreement with cultures. However, the interpretation of mNGS data should be combined with clinical data and conventional methods considering the lack of unified diagnostic criteria.

METAGENOMIC NEXT-GENERATION SEQUENCING DETECTS PATHOGENS IN ENDOPHTHALMITIS PATIENTS.

To investigate the utility of metagenomic next-generation sequencing (mNGS) in identifying the pathogens in endophthalmitis. In this prospective study, 36 cases of endophthalmitis were recruited. All patients received surgical treatment and intraocular drug lavage. The samples of vitreous or aqueous humor were extracted for mNGS and microbiological culture. The diagnostic performance of pathogens was compared between mNGS and culture. The positive rates of mNGS and culture were 88.89% (32/36) and 27.78% (10/36), respectively. There was a statistically significant difference between mNGS and culture (Chi-square = 27.657; P < 0.01). Staphylococcus epidermidis, Streptococcus pneumoniae, and Klebsiella pneumoniae were the most pathogenic bacteria in traumatic, postoperative, and endogenous endophthalmitis, respectively. The concordance of pathogen identified by mNGS and culture was 70% for culture-positive cases. Antibiotic resistance genes were identified in 9 cases. There was a marginal correlation between the final visual acuity and the microbial sequence read (Spearman correlation coefficient = 0.498; P = 0.05). Metagenomic next-generation sequencing has a higher positive rate of identifying pathogens in endophthalmitis than in culture. It can also provide information on antibiotic resistance and visual prognosis. However, caution must be taken when interpreting the results of mNGS because they may not be concordant with culture.

Metagenomic Next-Generation Sequencing of Cerebrospinal Fluid for the Diagnosis of Cerebral Aspergillosis.

Purpose: Cerebral aspergillosis (CA) is a rare but often fatal, difficult-to-diagnose, opportunistic infection. The utility of metagenomic next-generation sequencing (mNGS) for diagnosis of CA is unclear. We evaluated the usefulness of mNGS of the cerebrospinal fluid (CSF) for the diagnosis of CA.Methods: This prospective study involved seven consecutive patients with confirmed CA in whom CSF mNGS was performed. Serum (1→3)-β-D-glucan and galactomannan levels were determined, and histopathological examination and mNGS of the CSF were conducted. CSF specimens from three non-infected patients were used as positive controls.Results: mNGS of the CSF was positive in six of the seven confirmed CA cases (85.71% sensitivity). In the cryptococcal meningitis group (control), mNGS of the CSF was positive for Aspergillus in two patients (84.62% specificity). The positive likelihood ratio, negative likelihood ratio, and Youden’s index of mNGS for CA in the CSF were 5.565, 0.169, and 0.7, respectively. Among the six mNGS-positive cases, more than two Aspergillus species were found in four (4/6, 66.67%). In the positive controls, the addition of one A. fumigatus spore yielded a standardised species-specific read number (SDSSRN) of 25.45 by mNGS; the detection rate would be 0.98 if SDSSRN was 2.Conclusion: mNGS facilitates the diagnosis of CA and may reduce the need for cerebral biopsy in patients with suspected CA.Trial Registration Number: Chinese Clinical Trial Registry, ChiCTR1800020442.

Metagenomic Next-Generation Sequencing for the Diagnosis of Pneumocystis jirovecii Pneumonia in Non-HIV-Infected Patients: A Retrospective Study.

IntroductionThis study aimed to evaluate the utility of metagenomic next-generation sequencing (mNGS) for the diagnosis of Pneumocystis jirovecii pneumonia (PJP) in non-human immunodeficiency virus-infected patients.MethodsWe conducted a retrospective study. A total of 60 non-human immunodeficiency virus-infected PJP patients and 134 patients diagnosed with non-PJP pneumonia were included. P. jirovecii and other co-pathogens identified by mNGS in bronchoalveolar lavage fluid and/or blood samples were analyzed. Using clinical composite diagnosis as the reference standard, we compared the diagnostic performance of mNGS in PJP with conventional methods, including Gomori methenamine silver staining and serum (1,3)-β-d-glucan. Modifications of antimicrobial treatment for PJP patients after the report of mNGS results were also reviewed.ResultsmNGS reached a sensitivity of 100% in diagnosing PJP, which was remarkably higher than Gomori methenamine silver staining (25.0%) and serum (1,3)-β-d-glucan (67.4%). The specificity of mNGS (96.3%) significantly surpassed serum (1,3)-β-d-glucan (81.4%). Simultaneous mNGS of bronchoalveolar lavage fluid and blood samples was performed in 21 out of 60 PJP patients, and it showed a concordance rate of 100% in detecting P. jirovecii. Besides, mNGS showed good performance in identifying co-pathogens of PJP patients, among which cytomegalovirus and Epstein-Barr virus were most commonly seen. Initial antimicrobial treatment was modified in 71.7% of PJP patients after the report of mNGS results.ConclusionmNGS is a useful diagnostic tool with good performance for the diagnosis of PJP and the detection of co-pathogens. mNGS of bronchoalveolar lavage fluid and/or blood samples is suggested in patients with presumptive diagnosis of PJP. Blood samples may be a good alternative to bronchoalveolar lavage fluid for mNGS when bronchoscopic examination is not feasible.

Exploring the Clinical Utility of Metagenomic Next-Generation Sequencing in the Diagnosis of Pulmonary Infection.

IntroductionWe aimed to explore the real-world clinical application value and challenges of metagenomic next-generation sequencing (mNGS) for pulmonary infection diagnosis.MethodsWe retrospectively reviewed the results of mNGS and conventional tests from 140 hospitalized patients with suspected pulmonary infections from January 2019 to December 2020. The sample types included bronchoalveolar lavage fluid, lung tissue by transbronchial lung biopsy, pleural effusion, blood, and bronchial sputum. Apart from the mNGS reports that our patients received, an extra comprehensive and thorough literature search was conducted.ResultsSignificant differences were noticed in the positive detection rates of pathogens between mNGS and conventional diagnostic testing (115/140, 82.14% vs 50/140, 35.71%, P < 0.05). The percentage of mNGS-positive patients was significantly higher than that of conventional testing-positive patients with regard to bacterial detection (P < 0.01), but no significant differences were found with regard to fungal detection (P = 0.67). Significant statistical differences were found between mixed infection cases (15, 22.70%) and single infection cases (4, 7.84%) in terms of diabetes (P = 0.03). The most frequent pattern of mixed infection was bacteria and fungi mixed infection (40, 40/89 = 44.94%), followed by bacteria mixed infection (29, 29/89 = 32.58%). The sensitivity of mNGS in pulmonary infection diagnosis was much higher than that of conventional test (89.17% vs 50.00%; P < 0.01), but the specificity was the opposite (75.00% vs 81.82%; P > 0.05).ConclusionmNGS is a valuable tool for the detection of pulmonary infections, especially mixed pulmonary infections. The most common combinations we found were bacterial–fungal coinfection and bacterial–bacterial coinfection. Still, there are many challenges in the clinical application of mNGS in the diagnosis of pulmonary infections. There is still a lot of work to be done in interpreting the mNGS reports, because both clinical judgment and literature analysis strategy need to be refined.

Clinical usefulness of metagenomic next-generation sequencing for the diagnosis of central nervous system infection in people living with HIV

ObjectivesTo evaluate the clinical utility of metagenomic next-generation sequencing (mNGS) for the diagnosis of central nervous system (CNS) infection in people living with human immunodeficiency virus (PLWH) in a real-world situation. MethodsCerebrospinal fluid (CSF) was sent for mNGS for PLWH who tested negative on all conventional tests but were still suspected to have CNS infection. A retrospective analysis was undertaken of the results and the clinical effect of mNGS on this cohort. The final diagnosis was adjudicated by a panel discussion following hospital discharge when the results of all tests and patients’ responses to the empiric therapy were available. ResultsEighty-eight eligible PLWH, including 51 (58%) patients suspected of encephalitis and 34 (46.7%) patients suspected of meningitis, were included in the analysis. Sixty-eight (77.3%) patients were diagnosed with CNS infection, of which 50 were based on the pathogens identified by mNGS. The most common disease missed by mNGS was clinically suspected tuberculous meningitis, followed by clinically suspected non-tuberculous mycobacterial meningitis. The results from mNGS led to modification of treatment in 21 (23.9%) patients, and increased confidence in continuation of original therapy in 30 (34.1%) patients. During hospitalization, two (2.3%) patients died and 66 (75%) patients improved. ConclusionsmNGS of CSF is a useful tool for the diagnosis of CNS infection among PLWH. Further investigations are warranted to improve its sensitivity.