Improved Etiological Diagnosis of Nonresolving or Slowly Resolving Pneumonia Through Combined Endobronchial Ultrasound-Guided Biopsy and Metagenomic Sequencing

Which trial do we need? Plasma metagenomic next-generation sequencing to diagnose infections in patients with haematological malignancies and febrile neutropenia: proposal for a randomized-controlled trial

Endobronchial ultrasound

To observe the application of alveolar lavage fluid second-generation sequencing in diagnosing and guiding the treatment of patients with severe pneumonia with unidentified pathogenic bacteria and to provide novel ideas and methods for the effective clinical treatment of this disease. The clinical data of 80 patients with severe pneumonia included in our intensive care unit from June 2020 to June 2022 were analyzed and all patients had undergone metagenomic next-generation sequencing and traditional cultures (alveolar lavage fluid culture, sputum culture) to analyze the advantages of metagenomic next-generation sequencing in detecting pathogens in patients with severe pneumonia. The positive rate of metagenomic next-generation sequencing pathogen detection was higher than that of conventional culture. In patients who were negative for conventional culture and positive for metagenomic next-generation sequencing, metagenomic nextgeneration sequencing was able to further identify multiple pathogenic infections. In terms of pathogen distribution, metagenomic next-generation sequencing detected 71 bacterial, 39 fungal and 3 viral strains. In mixed infections, metagenomic next-generation sequencing yielded a higher rate of positive diagnosis. In addition, metagenomic next-generation sequencing had a higher pathogen detection rate in patients with combined underlying diseases and metagenomic next-generation sequencing could identify specific pathogenic infections especially in patients with combined immunocompromised conditions. Metagenomic next-generation sequencing could improve the detection rate of pathogenic microorganisms in patients with severe pneumonia and could be used as a complementary test for patients negative for conventional cultures. Metagenomic next-generation sequencing has advantages in the diagnosis of mixed infections and can identify multiple pathogenic infections simultaneously. Clinically, early application of metagenomic next-generation sequencing in patients with severe pneumonia is recommended for increased clinical benefit.

This study assessed the application of metagenomic next-generation sequencing in pathogen detection of periprosthetic joint infections. A total of 95 cases who previously had undergone hip and knee replacement undergoing revision from January 2018 to January 2021 were included in this study. Specimens of synovial fluid and deep-tissue were collected for culture and metagenomic next-generation sequencing, and patients were retrospectively categorized as infected or aseptic using the Musculoskeletal Infection Society criteria after revision surgery. The sensitivity, specificity, positive and negative predictive values were compared. A total of 36 cases had positive culture results and 59 cases had positive metagenomic next-generation sequencing results. Culture was positive in 34 infected cases (58.6%) and 2 aseptic cases (5.4%). Metagenomic next-generation sequencing was positive in 55 infected cases (94.8%) and 4 aseptic cases (10.8%). Five cases diagnosed with infection had other potential pathogens detected by metagenomic next-generation sequencing. Among the 24 culture-negative periprosthetic joint infections, metagenomic next-generation sequencing was able to identify potential pathogens in 21 cases (87.5%). From sampling to reporting, the average time needed for culture was 5.2 (95% CI 3.1–7.3) days, while that for metagenomic next-generation sequencing was 1.3 (95% CI 0.9–1.7) days. Metagenomic next-generation sequencing is more advantageous in pathogen detection of periprosthetic joint infection after total joint replacement, especially in patients with multiple infections or negative culture results.

Metagenomic next-generation sequencing offers an unbiased approach to identifying viral pathogens in cerebrospinal fluid of patients with meningoencephalitis of unknown etiology. In an 11-month case series, we investigated the use of cerebrospinal fluid metagenomic next-generation sequencing to diagnose viral infections among pediatric hospitalized patients presenting with encephalitis or meningoencephalitis of unknown etiology. Cerebrospinal fluid from patients with known enterovirus meningitis were included as positive controls. Cerebrospinal fluid from patients with primary intracranial hypertension were included to serve as controls without known infections. Cerebrospinal fluid metagenomic next-generation sequencing was performed for 37 patients. Among 27 patients with encephalitis or meningoencephalitis, 4 were later diagnosed with viral encephalitis, 6 had non-central nervous system infections with central nervous system manifestations, 6 had no positive diagnostic tests, and 11 were found to have a noninfectious diagnosis. Metagenomic next-generation sequencing identified West Nile virus (WNV) in the cerebrospinal fluid of 1 immunocompromised patient. Among the 4 patients with known enterovirus meningitis, metagenomic next-generation sequencing correctly identified enteroviruses and characterized the viral genotype. No viral sequences were detected in the cerebrospinal fluid of patients with primary intracranial hypertension. Metagenomic next-generation sequencing also identified sequences of nonpathogenic torque Teno virus in cerebrospinal fluid specimens from 13 patients. Our results showed viral detection by cerebrospinal fluid metagenomic next-generation sequencing only in 1 immunocompromised patient and did not offer a diagnostic advantage over conventional testing. Viral phylogenetic characterization by metagenomic next-generation sequencing could be used in epidemiologic investigations of some viral pathogens, such as enteroviruses. The finding of torque Teno viruses in cerebrospinal fluid by metagenomic next-generation sequencing is of unknown significance but may merit further exploration for a possible association with noninfectious central nervous system disorders.

BackgroundCommunity-acquired central nervous system infections (CA-CNS infections) have the characteristics of acute onset and rapid progression, and are associated with high levels of morbidity and mortality worldwide. However, there have been only limited studies on the etiology of this infections. Here, metagenomic next-generation sequencing (mNGS), a comprehensive diagnosis method, facilitated us to better understand the etiology of CA-CNS infections.MethodsWe conducted a single-center retrospective study between September 2018 and July 2021 in which 606 cerebrospinal fluid (CSF) samples were collected from suspected CNS infectious patients for mNGS testing, and all positive samples were included in this analysisResultsAfter the exclusion criteria, a total of 131 mNGS-positive samples were finally enrolled. Bacterial, viral, fungal, parasitic, specific pathogen and mixed infections were accounted for 32.82% (43/131), 13.74% (18/131), 0.76% (1/131), 2.29% (3/131) and 6.87% (9/131), respectively. A total of 41 different pathogens were identified, including 16 bacteria, 12 viruses, 10 fungi, and 1 parasite and 3 specific pathogens. The most frequent infecting pathogens are Epstein-Barr virus (n = 14), Herpes simplex virus 1 (n = 14), Mycobacterium tuberculosis (n = 13), Streptococcus pneumoniae (n = 13), and Cryptococcus neoformans (n = 8). Some difficult-to-diagnose pathogen infections were also detected by mNGS, such as Streptococcus suis, Pseudorabies virus, Bunyavirus, Orientia tsutsugamushi and Toxoplasma gondii.ConclusionIn this study, mNGS identified a wide variety of pathogens of CA-CNS infections and many of which could not be detected by conventional methods. Our data provide a better understanding of the etiology of CA-CNS infections and show that mNGS represents a comparative screening of CSF in an unbiased manner for a broad range of human pathogens.

Metagenomic next-generation sequencing (mNGS) offers an agnostic approach for emerging pathogen detection directly from clinical specimens. In contrast to targeted methods, mNGS also provides valuable information on the composition of the microbiome and might uncover coinfections that may associate with disease progression and impact prognosis. To evaluate the use of mNGS for detecting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and/or other infecting pathogens, we applied direct Oxford Nanopore long-read third-generation metatranscriptomic and metagenomic sequencing. Nasopharyngeal (NP) swab specimens from 50 patients under investigation for CoV disease 2019 (COVID-19) were sequenced, and the data were analyzed by the CosmosID bioinformatics platform. Further, we characterized coinfections and the microbiome associated with a four-point severity index. SARS-CoV-2 was identified in 77.5% (31/40) of samples positive by RT-PCR, correlating with lower cycle threshold (Ct) values and fewer days from symptom onset. At the time of sampling, possible bacterial or viral coinfections were detected in 12.5% of SARS-CoV-2-positive specimens. A decrease in microbial diversity was observed among COVID-19-confirmed patients (Shannon diversity index, P = 0.0082; Chao richness estimate, P = 0.0097; Simpson diversity index, P = 0.018), and differences in microbial communities were linked to disease severity (P = 0.022). Furthermore, statistically significant shifts in the microbiome were identified among SARS-CoV-2-positive and -negative patients, in the latter of whom a higher abundance of Propionibacteriaceae (P = 0.028) and a reduction in the abundance of Corynebacterium accolens (P = 0.025) were observed. Our study corroborates the growing evidence that increased SARS-CoV-2 RNA detection from NP swabs is associated with the early stages rather than the severity of COVID-19. Further, we demonstrate that SARS-CoV-2 causes a significant change in the respiratory microbiome. This work illustrates the utility of mNGS for the detection of SARS-CoV-2, for diagnosing coinfections without viral target enrichment or amplification, and for the analysis of the respiratory microbiome.IMPORTANCE SARS-CoV-2 has presented a rapidly accelerating global public health crisis. The ability to detect and analyze viral RNA from minimally invasive patient specimens is critical to the public health response. Metagenomic next-generation sequencing (mNGS) offers an opportunity to detect SARS-CoV-2 from nasopharyngeal (NP) swabs. This approach also provides information on the composition of the respiratory microbiome and its relationship to coinfections or the presence of other organisms that may impact SARS-CoV-2 disease progression and prognosis. Here, using direct Oxford Nanopore long-read third-generation metatranscriptomic and metagenomic sequencing of NP swab specimens from 50 patients under investigation for COVID-19, we detected SARS-CoV-2 sequences by applying the CosmosID bioinformatics platform. Further, we characterized coinfections and detected a decrease in the diversity of the microbiomes in these patients. Statistically significant shifts in the microbiome were identified among COVID-19-positive and -negative patients, in the latter of whom a higher abundance of Propionibacteriaceae and a reduction in the abundance of Corynebacterium accolens were observed. Our study also corroborates the growing evidence that increased SARS-CoV-2 RNA detection from NP swabs is associated with the early stages of disease rather than with severity of disease. This work illustrates the utility of mNGS for the detection and analysis of SARS-CoV-2 from NP swabs without viral target enrichment or amplification and for the analysis of the respiratory microbiome.

Cryptococcus neoformans osteomyelitis coupled with tuberculosis and tuberculous lymphadenitis, is a rare occurrence in clinical. Diagnostic challenges arise due to the clinical radiological similarity of this condition to other lung infections and the limited and sensitive nature of traditional approaches. Here, we present a case of co-infection diagnosed using Metagenomic Next-Generation Sequencing, highlighting the effectiveness of advanced genomic techniques in such complex scenarios. We present a case of a 67-year-old female infected with cryptococcal osteomyelitis and presented with swelling and pain in the right ankle. Following a biopsy of the right ankle joint, Metagenomic Next-Generation Sequencing (mNGS) of the biopsy tissue revealed Cryptococcus neoformans infection. Positive results for Cryptococcus capsular antigen and pathological findings confirmed the presence of Cryptococcus neoformans. The patient underwent surgical debridement, coupled with oral fluconazole treatment (300mg/day), leading to the resolution of symptoms. Cryptococcus neoformans is an uncommon cause of ankle infection. Metagenomic Next-Generation Sequencing (mNGS) serves as a valuable diagnostic tool, aiding clinicians in differentiating cryptococcal osteomyelitis from other atypical infections.

Vibrio cholerae is the causative agent of the human intestinal infectious disease cholera, which includes a variety of serogroups. However, there have been very few cases of hepatic space-occupying lesions associated with this infection. Currently, there are various methods for detecting this pathogen, including metagenomic sequencing, which enables quicker and more accurate identification. In this study, metagenomic sequencing is employed to accurately identify non-O1/O139 Vibrio cholerae infections by analyzing the genetic material present in clinical samples. A 75-year-old man presented with diarrhea and fever after consuming crabs. The initial treatment improved the diarrhea, but a liver abscess developed later. Magnetic resonance imaging (MRI) of the liver revealed a hepatic space-occupying lesion. Upon further investigation, a Gram-negative, rod-shaped bacterium was cultured from the patient's liver puncture fluid, and Vibrio cholerae was detected in the same fluid using metagenomic next-generation sequencing (mNGS). The pathogen was confirmed to be non-O1/non-O139 Vibrio cholerae (NOVC) using polymerase chain reaction (PCR). Following treatment with piperacillin/tazobactam sodium and moxifloxacin, the patient's body temperature returned to normal, the liver abscess improved significantly, and he was subsequently discharged from the hospital. This case study describes an elderly male patient with a hepatic space-occupying lesion. Multiple cultures of specimens failed to identify the underlying cause; however, advanced techniques such as mNGS and PCR confirmed an NOVC infection. This indicates that mNGS can serve as a valuable tool in diagnosing cases of unexplained liver infections. The use of mNGS is significant for detecting and clinically diagnosing infectious pathogens in patients with unexplained space-occupying lesions.

Clinical management and surveillance of the Enterobacter cloacae complex (ECC) face significant challenges due to inaccurate species identification and prolonged turnaround time for culture-based antimicrobial susceptibility testing (AST). To date, no studies have leveraged whole-genome sequencing (WGS) and metagenomic next-generation sequencing (mNGS) to develop a rapid AST prediction model for ECC. Here, a total of 1,054 ECC strain genomes with AST data were collected from a public database and a local hospital. The results of species identification between the average nucleotide identity (ANI)-based method on culture were compared, and machine learning was employed to identify resistance features for imipenem (IPM), meropenem (MEM), ciprofloxacin (CIP), levofloxacin (LEV), and trimethoprim-sulfamethoxazole (SXT). By referring to ANI-based species classification, culture-based methods showed a 74% misidentification rate for 1,054 ECC isolates. The antimicrobial resistance prediction model demonstrated good performance, with the area under the curve values of 91.25% (IPM), 89.69%, 88.17% (CIP), 91.01% (LEV), and 90.93% (SXT) respectively. Moreover, a combined WGS and mNGS approach was utilized and validated using 104 pediatric sputum specimens. Compared to culture-based AST, the overall accuracy of models exceeded 95%, especially achieving 100% for IPM and 98.80% for MEM, and the detection turnaround time was shortened by 69.64 h. Furthermore, it would enable early escalated therapy in 20.83% of cases, significantly improving patient management. This established WGS and mNGS-based AST prediction model addresses the limitations of traditional methods, offering a rapid, accurate, and clinically applicable tool for managing multidrug-resistant ECC infections.IMPORTANCEThe Enterobacter cloacae complex (ECC) poses a major challenge to clinical management due to difficulties in accurate species identification and the slow turnaround times of conventional culture-based antimicrobial susceptibility testing (AST). Current methods are often inefficient and prone to misidentification, leading to delayed or inappropriate treatment. This study introduces a novel approach that combines whole-genome sequencing (WGS) and metagenomic next-generation sequencing (mNGS) to develop a rapid and accurate AST prediction model for ECC. By leveraging machine learning to analyze WGS data from over 1,000 ECC isolates and validating the model with pediatric clinical specimens. The model achieved over 88% area under the curve accuracy for all antibiotics, demonstrated >95% accuracy in clinical validation, and reduced detection turnaround time by 69.64 h compared to traditional methods. The model has the potential to revolutionize ECC management by facilitating timely, targeted therapies and enhancing patient outcomes, especially in the context of multidrug-resistant infections.

Treponema phagedenis, a human commensal spirochete, has been reported world-wide as a key factor in the pathogenesis of bovine digital dermatitis. Here we report a case of T. phagedenis sequence detection in the cerebrospinal fluid (CSF) of a patient. The patient was diagnosed with neurosyphilis, and T. phagedenis was detected as the only microorganism in his CSF by metagenomic sequencing. The patient went through a round of penicillin therapy previously (2.4 million units of Benzathine Penicillin intramuscularly once a week for three weeks) that did not resolve the symptoms; after the diagnosis of neurosyphilis he was treated with Penicillin G Sodium 4.0 million units q4h intravenous for 14 days then his symptoms resolved. To the best of our knowledge, T. phagedenis has never been reported to be detected in a human’s CSF before. This was also the first time it was detected by metagenomic next-generation sequencing. We propose that more etiological tests should be performed including culture and sequencing for more patients with syphilis, which will contribute to a deeper understanding of the pathogenicity of the spirochete.

Background Metagenomic next-generation sequencing (mNGS) is a new technology that allows for unbiased detection of pathogens. However, there are few reports on mNGS of lung biopsy tissues for pulmonary infection diagnosis. In addition, radial endobronchial ultrasound (R-EBUS) is widely used to detect peripheral pulmonary lesions (PPLs), but it is rarely used in the diagnosis of peripheral lung infection. Objective The present study aims to evaluate the combined application of R-EBUS-guided transbronchial lung biopsy (TBLB) and mNGS for the diagnosis of peripheral pulmonary infectious lesions. Methods From July 2018 to April 2019, 121 patients from Tianjin Medical University General Hospital diagnosed with PPLs and lung infection were enrolled in this prospective randomized study . Once the lesion was located, either TBLB or R-EBUS-guided-TBLB was performed in randomly selected patients, and mNGS was applied for pathogen detection in lung biopsy tissues. The results of mNGS were compared between the TBLB group and R-EBUS-guided TBLB group. In addition, the clinical characteristics and EBUS images from 61 patients receiving bronchoscopy for peripheral lung infectious detection were analyzed and compared with the results of mNGS. Results The positivity rate of mNGS in R-EBUS-guided TBLB was (78.7%, 48/61) that was significantly higher than (60.0%, 36/60) in the TBLB group. Difference in the position of R-EBUS probe and image characteristics of peripheral lung infectious lesions affected the positivity rate of mNGS. Tissue collected by R-EBUS within the lesion produced higher positivity rate than samples collected adjacent to the lesion (P=0.030, odds ratio 17.742; 95% confidence interval, from 1.325 to 237.645). Anechoic areas and luminant areas of ultrasonic image characteristics were correlated with lower positivity rate of mNGS (respectively, P=0.019, odds ratio 17.878; 95% confidence interval, from 1.595 to 200.399; P=0.042, odds ratio 16.745; 95% confidence interval, from 1.106 to 253.479). Conclusions R-EBUS-guided TBLB is a safe and effective technique in the diagnosis of peripheral lung infectious lesions. R-EBUS significantly facilitates the accurate insertion of bronchoscope into the lesions, which improves positivity rate of mNGS analysis in pathogen detection. The R-EBUS probe position within lesion produced a higher positivity rate of mNGS analysis. Nevertheless, the presence of anechoic and luminant areas on ultrasonic image was correlated with poor mNGS positivity rate.

The aim of this study was to compare metagenomic next-generation sequencing (mNGS) with other methods, including Xpert MTB/RIF, Mycobacterium tuberculosis (MTB) culture, and acid-fast bacillus (AFB) staining in the diagnosis of pulmonary tuberculosis (PTB) using bronchoalveolar lavage fluid (BALF). The data of 186 patients with suspected PTB were retrospectively collected from January 2020 to May 2021 at Tongji Hospital. BALF samples were collected from all patients and analyzed using AFB staining, MTB culture, Xpert MTB/RIF, and mNGS. Of the 186 patients, 38 patients were ultimately diagnosed as PTB. Metagenomic next-generation sequencing exhibited a sensitivity of 78.95%, which was higher than AFB staining (27.59%) and MTB culture (44.12%) but similar to Xpert MTB/RIF (72.73%). Utilization of combined methods demonstrates improvement for PTB diagnosis. In support of this, the area under the receiver operating characteristic curve for the combination of mNGS and MTB culture (0.933, 95% CI: 0.871, 0.995) was larger than those of mNGS, Xpert MTB/RIF, MTB culture, and the combination of Xpert MTB/RIF and MTB culture. The sensitivity of mNGS in the diagnosis of PTB using BALF specimen is similar to Xpert MTB/RIF.Metagenomic next-generation sequencing in combination with MTB culture may further improve the diagnosis of pulmonary tuberculosis.

Background: Radial probe endobronchial ultrasound-guided transbronchial lung biopsy (RP-EBUS-TBLB) is widely used for diagnosis of peripheral lung lesions (PLLs). To date, there have been no reports regarding the clinical outcomes of RP-EBUS-TBLB for PLLs in patients with idiopathic pulmonary fibrosis (IPF). Objectives: This study was performed between October 2017 and December 2019 to identify the safety and diagnostic performance of RP-EBUS-TBLB in IPF patients. Methods: Patients were divided into the usual interstitial pneumonia (UIP) group (n = 39, 4%), the probable UIP group (n = 12, 1%), and the noninterstitial lung disease (non-ILD) group (n = 903, 95%). Results: The diagnostic yield was significantly lower in the UIP group than in the non-ILD group (62% vs. 76%; p = 0.042), but there were no significant differences between the UIP and probable UIP groups (62% vs. 83%; p = 0.293) or the probable UIP and non-ILD groups (83% vs. 76%; p = 0.741). Multivariate logistic analysis showed that the mean diameter of PLLs, positive bronchus sign on CT, and “within the lesion” status on EBUS were independently associated with success of the procedure. Especially, the presence of the UIP pattern on CT (OR, 0.385; 95% CI: 0.172–0.863; p = 0.020) was independently associated with failed diagnosis. Among patients with UIP, “within the lesion” status on EBUS (OR, 25.432; 95% CI: 2.321–278.666; p = 0.008) was shown to be a factor contributing to a successful diagnosis. Overall, there were no significant differences in complication rates among the 3 study groups. Conclusion: RP-EBUS-TBLB can be performed safely with an acceptable diagnostic yield, even in patients with IPF.

Objective To evaluate the value of nanopore targeted sequencing in diagnosing pneumonia pathogens. Methods This large-scale multicentre prospective study performed in 8 hospitals across China from April to October 2022. Hospitalised patients with a diagnosis of pneumonia at admission were included. Complete clinical data were collected, and bronchoalveolar lavage fluid were obtained from each patient. These samples underwent simultaneous testing using conventional microbial testing, metagenomic next-generation sequencing, and nanopore targeted sequencing. Results A total of 218 patients were included. Among the 168 cases of pulmonary infection, 246 strains of pathogens were confirmed. Nanopore targeted sequencing outperformed conventional microbial testing, identifying more pathogens with a sensitivity increase of 47.9% (77.2% vs. 29.3%). Metagenomic next-generation sequencing had a sensitivity of 82.9%. Total of 70.1% patients had consistent results in both metagenomic next-generation sequencing and nanopore targeted sequencing. Nanopore targeted sequencing exhibited significantly higher sensitivity in detecting Pneumocystis jiroveci, cytomegalovirus, Mycobacterium tuberculosis, Nontuberculous mycobacteria, Streptococcus pneumoniae, and Mycoplasma pneumoniae compared to conventional microbial testing. However, metagenomic next-generation sequencing demonstrated higher sensitivity than nanopore targeted sequencing for Aspergillus (88.5% vs. 53.8%). Regarding the detection of co-infections, nanopore targeted sequencing displayed significantly higher sensitivity than conventional microbial testing (76.7% vs. 28.7%) and was on par with metagenomic next-generation sequencing (76.7% vs. 82.9%). Conclusion Nanopore targeted sequencing performs equally well as metagenomic next-generation sequencing in bronchoalveolar lavage fluid for pathogen diagnosis in pneumonia, both methods showing higher sensitivity than conventional microbial testing. Nanopore targeted sequencing can be considered a reliable method for diagnosing pathogens in pneumonia.