Metagenomic next-generation sequencing to identify pathogens and cancer in lung biopsy tissue
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ABSTRACT: Background
Lung biopsy tissue samples can be used for infection detection and cancer diagnosis. Metagenomic next-generation sequencing (mNGS) has the potential to further improve diagnosis. Methods
From July 2018 to May 2020, lung biopsy samples of 133 patients with suspected pulmonary infection or abnormal imaging findings were collected and subjected to clinical microbiological testing, Illumina and Nanopore sequencing to identify pathogens. The neural networks were pretrained by extracting features of human reads from 2,095 metagenomic next-generation sequencing results, and the human reads of lung biopsy samples were entered into the validated pipeline to predict the risk of cancer. Findings
Based on the pathogen-cancer detection pipeline, the Illumina platform showed 77·6% sensitivity and 97·6% specificity compared to the composite reference standard for infection diagnosis. However, the Nanopore platform showed 34·7% sensitivity and 98·7% specificity. mNGS identified more fungi, which was confirmed by subsequent pathological examination. M. tuberculosis complex was weakly detected. For cancer detection, compared with histology, the Illumina platform showed 83·7% sensitivity and 97·6% specificity, diagnosing an additional 36 cancer patients, of whom half had abnormal imaging findings (pulmonary shadow, space-occupying lesions, or nodules). Interpretation
For the first time, we have established a pipeline to simultaneously detect pathogens and cancer based on Illumina sequencing of lung biopsy tissue. This pipeline efficiently diagnosed cancer in patients with abnormal imaging findings. Funding
This work was supported by the National Key Research and Development Program of China and National Natural Science Foundation of China.
SUBMITTER: Guo Y
PROVIDER: S-EPMC8554462 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:ObjectivesClinicians often face the challenge of differentially diagnosing febrile patients who are suspected of infectious diseases, since the clinical manifestations of infection and cancer may overlap. A single test that can detect both pathogens and tumor could provide timely and accurate diagnostic clues to aid the treatment and management of these patients.MethodsWe enrolled eight patients to evaluate the utility of metagenomic Next-Generation Sequencing for simultaneously detecting pathogens and neoplasms using body fluids and tissue samples. Patients were selected by the following criteria: 1) Tumor was not considered upon hospitalization, but mNGS testing indicated neoplasm; 2) Tumor was not excluded, but microbial infection was primarily suspected according to initial clinical assessment.ResultsWe detected potential pathogens in five patients, three of whom had progressed into critical infections. Moreover, abnormal chromosomal copy numbers were identified in all patients that indicated presence of neoplasms, which were pathologically confirmed.ConclusionsAlthough copy number variations do not render a definitive cancer diagnosis, it can prompt clinicians to conduct more focused diagnostic testing for cancer, potentially saving time and cost. As a result, integrating copy number analysis with pathogen detection in mNGS may help establish rapid and accurate diagnosis for febrile patients.
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Project description:BACKGROUND: The introduction of benchtop sequencers has made adoption of whole genome sequencing possible for a broader community of researchers than ever before. Concurrently, metagenomic sequencing (MGS) is rapidly emerging as a tool for interrogating complex samples that defy conventional analyses. In addition, next-generation sequencers are increasingly being used in clinical or related settings, for instance to track outbreaks. However, information regarding the analytical sensitivity or limit of detection (LoD) of benchtop sequencers is currently lacking. Furthermore, the specificity of sequence information at or near the LoD is unknown. RESULTS: In the present study, we assess the ability of three next-generation sequencing platforms to identify a pathogen (viral or bacterial) present in low titers in a clinically relevant sample (blood). Our results indicate that the Roche-454 Titanium platform is capable of detecting Dengue virus at titers as low as 1X102.5 pfu/mL, corresponding to an estimated 5.4X104 genome copies/ml maximum. The increased throughput of the benchtop sequencers, the Ion Torrent PGM and Illumina MiSeq platforms, enabled detection of viral genomes at concentrations as low as 1X104 genome copies/mL. Platform-specific biases were evident in sequence read distributions as well as viral genome coverage. For bacterial samples, only the MiSeq platform was able to provide sequencing reads that could be unambiguously classified as originating from Bacillus anthracis. CONCLUSION: The analytical sensitivity of all three platforms approaches that of standard qPCR assays. Although all platforms were able to detect pathogens at the levels tested, there were several noteworthy differences. The Roche-454 Titanium platform produced consistently longer reads, even when compared with the latest chemistry updates for the PGM platform. The MiSeq platform produced consistently greater depth and breadth of coverage, while the Ion Torrent was unequaled for speed of sequencing. None of the platforms were able to verify a single nucleotide polymorphism responsible for antiviral resistance in an Influenza A strain isolated from the 2009 H1N1 pandemic. Overall, the benchtop platforms perform well for identification of pathogens from a representative clinical sample. However, unlike identification, characterization of pathogens is likely to require higher titers, multiple libraries and/or multiple sequencing runs.
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Project description: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.
| S-EPMC7165338 | biostudies-literature
Project description:PurposeThis study aims to evaluate the diagnostic application and performance of the metagenomic next-generation sequencing (mNGS) in patients suspected of local pulmonary infection by comparing it to the traditional pathogen detection methods in lung tissue specimens obtained by a computerized tomography-guided biopsy (CT-guided biopsy).MethodsWe retrospectively reviewed patients, admitted to the First Affiliated Hospital of Wenzhou Medical University, China from May 2018 to December 2020, who were suspected of local pulmonary infection. All cases received a CT-guided lung biopsy, tissue samples were sent both for conventional examinations (CE) and mNGS tests. The sensitivity and specificity of the two diagnostic approaches were compared.Results106 patients enrolled, 76 patients were diagnosed with a pulmonary infection. Among 49 patients with identified pathogens, CE confirmed pathogenic infections in 32 cases. Mycobacterium spp. and fungi accounted for 37.5% (12/32) and 28.1% (9/32), respectively, with bacteria 34.4% (11/32). The mNGS examination detected extra pathogenic microorganisms in 22 patients that were consistent with the patients' clinical and radiographic pictures. The sensitivity of mNGS was 53.9% vs. 42.1% for the CE, while the specificity was 56.7% versus 96.7%. For detection rate, mNGS was significantly superior to CE in bacterial (96.3% vs. 40.7%, p < 0.05), and mixed infections (100% vs. 50%, p < 0.05), but inferior to CE in fungal (60% vs. 90%, p > 0.05) and Mycobacterium spp. infections (66.7% vs. 100%, p > 0.05) with no significant difference. Among 31 cases diagnosed with lung abscess, the diagnostic performance of the detection rate was 67.7% (21/31) in favour of mNGS compared to 29.0% (9/31) for CE (p < 0.05). Most polymicrobial infections were induced by anaerobic species that coexisted with Streptococcus constellatus. And Klebsiella pneumoniae was the most common isolated monomicrobial infection.ConclusionsThe most commonly detected causative pathogens for local pulmonary infections were bacteria, Mycobacterium spp. and fungi. Compared with the CE, the advantages of mNGS in the pathogens detection lie in the discovery of bacterial and mixed infections, as well as in the detection of lung abscess. Conversely, mNGS is not good enough to be recommendable for the detection of Mycobacterium spp. and fungi.
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Project description:Tick-borne diseases are a major public health concern in Mongolia. Nomadic pastoralists, which make up ~ 26% of Mongolia's population, are at an increased risk of both tick bite exposure and economic loss associated with clinical disease in herds. This study sought to further characterize tick-borne pathogens present in Dermacentor ticks (n = 1,773) sampled in 2019 from 15 of Mongolia's 21 aimags (provinces). The ticks were morphologically identified and sorted into 377 pools which were then screened using Next-Generation Sequencing paired with confirmatory PCR and DNA sequence analysis. Rickettsia spp. were detected in 88.33% of pools, while Anaplasma spp. and Bartonella spp. were detected in 3.18 and 0.79% of pools, respectively. Khentii had the highest infection rate for Rickettsia spp. (76.61%; CI: 34.65-94.79%), while Arkhangai had the highest infection rate for Anaplasma spp. (7.79%; CI:4.04-13.72%). The exclusive detection of Anaplasma spp. in tick pools collected from livestock supports previous work in this area that suggests livestock play a significant role in disease maintenance. The detection of Anaplasma, Bartonella, and Rickettsia demonstrates a heightened risk for infection throughout Mongolia, with this study, to our knowledge, documenting the first detection of Bartonella melophagi in ticks collected in Mongolia. Further research deploying NGS methods is needed to characterize tick-borne pathogens in other endemic tick species found in Mongolia, including Hyalomma asiaticum and Ixodes persulcatus.
| S-EPMC9399792 | biostudies-literature
Project description:BackgroundMetagenomic next-generation sequencing (mNGS) technology has been widely used to diagnose various infections. Based on the most common pathogen profiles, targeted mNGS (tNGS) using multiplex PCR has been developed to detect pathogens with predesigned primers in the panel, significantly improving sensitivity and reducing economic burden on patients. However, there are few studies on summarizing pathogen profiles of pulmonary infections in immunocompetent and immunocompromised patients in Jilin Province of China on large scale.MethodsFrom January 2021 to December 2023, bronchoalveolar lavage fluid (BALF) or sputum samples from 546 immunocompetent and immunocompromised patients with suspected community-acquired pneumonia were collected. Pathogen profiles in those patients on whom mNGS was performed were summarized. Additionally, we also evaluated the performance of tNGS in diagnosing pulmonary infections.ResultsCombined with results of mNGS and culture, we found that the most common bacterial pathogens were Pseudomonas aeruginosa, Klebsiella pneumoniae, and Acinetobacter baumannii in both immunocompromised and immunocompetent patients with high detection rates of Staphylococcus aureus and Enterococcus faecium, respectively. For fungal pathogens, Pneumocystis jirovecii was commonly detected in patients, while fungal infections in immunocompetent patients were mainly caused by Candida albicans. Most of viral infections in patients were caused by Human betaherpesvirus 5 and Human gammaherpesvirus 4. It is worth noting that, compared with immunocompetent patients (34.9%, 76/218), more mixed infections were found in immunocompromised patients (37.8%, 14/37). Additionally, taking final comprehensive clinical diagnoses as reference standard, total coincidence rate of BALF tNGS (81.4%, 48/59) was much higher than that of BALF mNGS (40.0%, 112/280).ConclusionsOur findings supplemented and classified the pathogen profiles of pulmonary infections in immunocompetent and immunocompromised patients in Jilin Province of China. Most importantly, our findings can accelerate the development and design of tNGS specifically used for regional pulmonary infections.
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