scholarly journals The microbiological diagnostic performance of metagenomic next-generation sequencing in patients with sepsis

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Di Ren ◽  
Chao Ren ◽  
Renqi Yao ◽  
Lin Zhang ◽  
Xiaomin Liang ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Pathogen Detection ◽  
Final Analysis ◽  
Data Interpretation ◽  
Lavage Fluid ◽  
Microbial Culture ◽  
Next Generation ◽  
Multiple Pathogens ◽  
Generation Sequencing

Abstract Background In this study, we aimed to perform a comprehensive analysis on the metagenomic next-generation sequencing for the etiological diagnosis of septic patients, and further to establish optimal read values for detecting common pathogens. Methods In this single-center retrospective study, septic patients who underwent pathogen detection by both microbial culture and metagenomic next-generation sequencing in the intensive care unit of the Second People’s Hospital of Shenzhen from June 24, 2015, to October 20, 2019, were included. Results A total of 193 patients with 305 detected specimens were included in the final analysis. The results of metagenomic next-generation sequencing showed significantly higher positive rates in samples from disparate loci, including blood, bronchoalveolar lavage fluid, and cerebrospinal fluid, as well as in the determination of various pathogens. The optimal diagnostic reads were 2893, 1825.5, and 892.5 for Acinetobacter baumannii, Pseudomonas aeruginosa, and Klebsiella pneumoniae, respectively. Conclusions The metagenomic next-generation sequencing is capable of identifying multiple pathogens in specimens from septic patients, and shows significantly higher positive rates than culture-based diagnostics. The optimal diagnostic reads for frequently detected microbes might be useful for the clinical application of metagenomic next-generation sequencing in terms of timely and accurately determining etiological pathogens for suspected and confirmed cases of sepsis due to well-performed data interpretation.

scholarly journals The Application of Metagenomic Next-Generation Sequencing in Detection of Pathogen in Bronchoalveolar Lavage Fluid and Sputum Samples of Patients with Pulmonary Infection

2021 ◽  
Vol 2021 ◽  
pp. 1-6
Author(s):  
Wanghui Shi ◽  
Shanshan Zhu
Keyword(s):  
Next Generation Sequencing ◽  
Bronchoalveolar Lavage ◽  
Pathogen Detection ◽  
Bronchoalveolar Lavage Fluid ◽  
Pulmonary Infection ◽  
Lavage Fluid ◽  
Microbial Culture ◽  
Next Generation ◽  
Two Samples ◽  
Generation Sequencing

Objective. To uncover the application value of metagenomic next-generation sequencing (mNGS) in the detection of pathogen in bronchoalveolar lavage fluid (BALF) and sputum samples. Methods. Totally, 32 patients with pulmonary infection were included. Pathogens in BALF and sputum samples were tested simultaneously by routine microbial culture and mNGS. Main infected pathogens (bacteria, fungi, and viruses) and their distribution in BALF and sputum samples were analyzed. Moreover, the diagnostic performance of mNGS in paired BALF and sputum samples was assessed. Results. The pathogen culture results were positive in 9 patients and negative in 13 patients. No statistical differences were recorded on the sensitivity (78.94% vs. 63.15%, p = 0.283 ) and specificity (62.50% vs. 75.00%, p = 0.375 ) of mNGS diagnosis in bacteria and fungus in two types of samples. As shown in mNGS detection, 10 patients’ two samples were both positive, 13 patients’ two samples were both negative, 7 patients were only positive in BALF samples, and 2 patients’ sputum samples were positive. Main viruses mNGS detected were EB virus, human adenovirus 5, herpes simplex virus type 1, and human cytomegalovirus. Kappa consensus analysis indicated that mNGS showed significant consistency in detecting pathogens in two samples, no matter bacteria ( p < 0.001 ), fungi ( p = 0.026 ), or viruses ( p = 0.008 ). Conclusion. mNGS showed no statistical differences in sensitivity and specificity of pathogen detection in BALF and sputum samples. Under certain conditions, sputum samples might be more suitable for pathogen detection because of invasiveness of BALF samples.

scholarly journals A Comparison of Blood Pathogen Detection Among Droplet Digital PCR, Metagenomic Next-Generation Sequencing, and Blood Culture in Critically Ill Patients With Suspected Bloodstream Infections

2021 ◽  
Vol 12 ◽  
Author(s):  
Bangchuan Hu ◽  
Yue Tao ◽  
Ziqiang Shao ◽  
Yang Zheng ◽  
Run Zhang ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Blood Culture ◽  
Critically Ill ◽  
Critically Ill Patients ◽  
Pathogen Detection ◽  
Bloodstream Infections ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Next Generation ◽  
Generation Sequencing

Metagenomic next-generation sequencing (mNGS) and droplet digital PCR (ddPCR) have recently demonstrated a great potential for pathogen detection. However, few studies have been undertaken to compare these two nucleic acid detection methods for identifying pathogens in patients with bloodstream infections (BSIs). This prospective study was thus conducted to compare these two methods for diagnostic applications in a clinical setting for critically ill patients with suspected BSIs. Upon suspicion of BSIs, whole blood samples were simultaneously drawn for ddPCR covering 20 common isolated pathogens and four antimicrobial resistance (AMR) genes, mNGS, and blood culture. Then, a head-to-head comparison was performed between ddPCR and mNGS. A total of 60 episodes of suspected BSIs were investigated in 45 critically ill patients, and ddPCR was positive in 50 (83.3%), mNGS in 41 (68.3%, not including viruses), and blood culture in 10 (16.7%) episodes. Of the 10 positive blood cultures, nine were concordantly identified by both mNGS and ddPCR methods. The head-to-head comparison showed that ddPCR was more rapid (~4 h vs. ~2 days) and sensitive (88 vs. 53 detectable pathogens) than mNGS within the detection range of ddPCR, while mNGS detected a broader range of pathogens (126 vs. 88 detectable pathogens, including viruses) than ddPCR. In addition, a total of 17 AMR genes, including 14 blaKPC and 3 mecA genes, were exclusively identified by ddPCR. Based on their respective limitations and strengths, the ddPCR method is more useful for rapid detection of common isolated pathogens as well as AMR genes in critically ill patients with suspected BSI, whereas mNGS testing is more appropriate for the diagnosis of BSI where classic microbiological or molecular diagnostic approaches fail to identify causative pathogens.

A Benchmark of Structural Variant Analysis Tools for Next Generation Sequencing Data

2013 ◽  
Vol 2 (4) ◽  
pp. 86-98
Author(s):  
Chatzinikolaou Panagiotis ◽  
Makris Christos ◽  
Dimitrios Vlachakis ◽  
Sophia Kossida
Keyword(s):  
Next Generation Sequencing ◽  
Level Structure ◽  
Atomic Level ◽  
Protein Sequencing ◽  
Next Generation Sequencing Data ◽  
Next Generation ◽  
Sequencing Data ◽  
Variant Analysis ◽  
Generation Sequencing

In language of genetics and biochemistry, sequencing is the determination of an unbranched biopolymer's primary structure. A sequence is a symbolic linear depiction, result of sequencing. This sequence is a succinct summary of the most of the sequenced molecule's atomic-level structure. (Most known is DNA-sequencing, RNA-sequencing, Protein-sequencing and Next-Generation-sequencing)

scholarly journals Metagenomic analysis using next-generation sequencing of pathogens in bronchoalveolar lavage fluid from pediatric patients with respiratory failure

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Suguru Takeuchi ◽  
Jun-ichi Kawada ◽  
Kazuhiro Horiba ◽  
Yusuke Okuno ◽  
Toshihiko Okumura ◽  
...  
Keyword(s):  
Respiratory Failure ◽  
Next Generation Sequencing ◽  
Bronchoalveolar Lavage ◽  
Bronchoalveolar Lavage Fluid ◽  
Pediatric Patients ◽  
Respiratory Infections ◽  
Sequence Data ◽  
Lavage Fluid ◽  
Next Generation ◽  
Generation Sequencing

Abstract Next-generation sequencing (NGS) has been applied in the field of infectious diseases. Bronchoalveolar lavage fluid (BALF) is considered a sterile type of specimen that is suitable for detecting pathogens of respiratory infections. The aim of this study was to comprehensively identify causative pathogens using NGS in BALF samples from immunocompetent pediatric patients with respiratory failure. Ten patients hospitalized with respiratory failure were included. BALF samples obtained in the acute phase were used to prepare DNA- and RNA-sequencing libraries. The libraries were sequenced on MiSeq, and the sequence data were analyzed using metagenome analysis tools. A mean of 2,041,216 total reads were sequenced for each library. Significant bacterial or viral sequencing reads were detected in eight of the 10 patients. Furthermore, candidate pathogens were detected in three patients in whom etiologic agents were not identified by conventional methods. The complete genome of enterovirus D68 was identified in two patients, and phylogenetic analysis suggested that both strains belong to subclade B3, which is an epidemic strain that has spread worldwide in recent years. Our results suggest that NGS can be applied for comprehensive molecular diagnostics as well as surveillance of pathogens in BALF from patients with respiratory infection.

scholarly journals Depletion of Human DNA in Spiked Clinical Specimens for Improvement of Sensitivity of Pathogen Detection by Next-Generation Sequencing

2016 ◽  
Vol 54 (4) ◽  
pp. 919-927 ◽  
Author(s):  
Mohammad R. Hasan ◽  
Arun Rawat ◽  
Patrick Tang ◽  
Puthen V. Jithesh ◽  
Eva Thomas ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Pathogen Detection ◽  
Clinical Samples ◽  
Metagenomic Sequencing ◽  
Next Generation ◽  
Simple Method ◽  
Clinical Specimens ◽  
Human Dna ◽  
Pathogen Dna ◽  
Generation Sequencing

Next-generation sequencing (NGS) technology has shown promise for the detection of human pathogens from clinical samples. However, one of the major obstacles to the use of NGS in diagnostic microbiology is the low ratio of pathogen DNA to human DNA in most clinical specimens. In this study, we aimed to develop a specimen-processing protocol to remove human DNA and enrich specimens for bacterial and viral DNA for shotgun metagenomic sequencing. Cerebrospinal fluid (CSF) and nasopharyngeal aspirate (NPA) specimens, spiked with control bacterial and viral pathogens, were processed using either a commercially available kit (MolYsis) or various detergents followed by DNase prior to the extraction of DNA. Relative quantities of human DNA and pathogen DNA were determined by real-time PCR. The MolYsis kit did not improve the pathogen-to-human DNA ratio, but significant reductions (>95%;P< 0.001) in human DNA with minimal effect on pathogen DNA were achieved in samples that were treated with 0.025% saponin, a nonionic surfactant. Specimen preprocessing significantly decreased NGS reads mapped to the human genome (P< 0.05) and improved the sensitivity of pathogen detection (P< 0.01), with a 20- to 650-fold increase in the ratio of microbial reads to human reads. Preprocessing also permitted the detection of pathogens that were undetectable in the unprocessed samples. Our results demonstrate a simple method for the reduction of background human DNA for metagenomic detection for a broad range of pathogens in clinical samples.

scholarly journals Next-Generation Sequencing of HIV-1 RNA Genomes: Determination of Error Rates and Minimizing Artificial Recombination

PLoS ONE ◽  
2013 ◽  
Vol 8 (9) ◽  
pp. e74249 ◽  
Author(s):  
Francesca Di Giallonardo ◽  
Osvaldo Zagordi ◽  
Yannick Duport ◽  
Christine Leemann ◽  
Beda Joos ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Error Rates ◽  
Next Generation ◽  
Generation Sequencing ◽  
Hiv 1

scholarly journals Gestational Psittacosis With Secondary Hemophagocytic Syndrome: A Case Report and Literature Review

2021 ◽  
Vol 8 ◽  
Author(s):  
Li Sun ◽  
Pulin Li ◽  
Bo Pang ◽  
Peipei Wu ◽  
Ran Wang
Keyword(s):  
Bone Marrow ◽  
Next Generation Sequencing ◽  
Hemophagocytic Syndrome ◽  
Nk Cell ◽  
Cell Activity ◽  
Lung Infection ◽  
Next Generation ◽  
Nk Cell Activity ◽  
Generation Sequencing

Gestational psittacosis and hemophagocytic syndrome (HPS) are rare clinical diseases. In this article, a case of gestational psittacosis concomitant with secondary HPS was reported. An analysis was performed on the clinical characteristics, signs, laboratory findings, progression, diagnosis, and treatment of a patient with gestational psittacosis concomitant with secondary HPS. Besides, the literature with respect to this disease was reviewed. This patient was definitively diagnosed through metagenomic next-generation sequencing techniques, bone marrow puncture and smear examination, and the determination of sCD25 level and natural killer (NK) cell activity. Anti-infectives such as doxycycline and etoposide combined with hormone chemotherapy achieved significant improvement in cough and expectoration, a return to normal temperature, and a significant improvement in oxygenation index. In addition, chest computed tomography revealed obvious absorption of lung lesions and a return of NK cell activity and sCD25 levels to normal ranges. Chlamydia psittaci pneumonia requires a clear determination of etiology, while HPS requires bone marrow puncture and smear examination, together with the determination of sCD25 level and NK cell activity in the blood. The findings of this study suggest that metagenomic next-generation sequencing is an effective instrument in clearly identifying pathogens that cause lung infection. Clinicians should consider atypical pathogens of lung infection in patients with poor response to empirical anti-infectives, and strive to design an effective treatment strategy as per an accurate diagnosis based on the etiology. As for patients suffering from long-term high fever and poor temperature control after broad-spectrum antibiotic treatment, non-infectious fever should be taken into account. A rapid and clear diagnosis would significantly improve patient prognosis.

scholarly journals Actinomyces as a Cause of Unexplained Fever Diagnosed by Next-Generation Sequencing: Case Report and Review

2021 ◽  
Author(s):  
Xiu Huang ◽  
Na Du ◽  
jiaxue He ◽  
yujun Du
Keyword(s):  
Next Generation Sequencing ◽  
Laboratory Data ◽  
Lavage Fluid ◽  
Next Generation ◽  
Intermittent Fever ◽  
Thoracic Actinomycosis ◽  
Sputum Production ◽  
Unexplained Fever ◽  
Generation Sequencing

Abstract Background: Actinomycosis is a rare cause of unexplained origin fever (FUO). The NGS, particularly in cases of FUO in conventional microbiology, is a promising and advantageous technology for earlier diagnosis and more success. It gives new insights in the management of FUO, especially in infectious diseases.Case presentation: We present a case of a 71-year-old woman who had FUO that the etiology was endobronchial actinomycosis eventually diagnosed by next-generation sequencing of bronchoalveolar lavage fluid. She was admitted to the our department with intermittent fever up to 40.0℃, chills, cough with white sputum, chest distress and slightly dyspnea over one month. According to special clinical course and laboratory data of the patient, she was given intravenous sulbenicillin sodium for 11 days and oral amoxicillin at 500 mg every 8 h for another 1 month. When she returned for follow-up, she reported a notable improvement in her cough, dyspnea, sputum production and fever that was completely absent.Conclusion: In obscure causes of FUO, clinicians should consider the possibility of thoracic actinomycosis and actively use next-generation sequencing to help diagnose.

scholarly journals A Retrospective Paired Comparison Between Untargeted Next Generation Sequencing and Conventional Microbiology Tests With Wisely Chosen Metagenomic Sequencing Positive Criteria

2021 ◽  
Vol 8 ◽  
Author(s):  
Hanyu Qin ◽  
Jinmin Peng ◽  
Ling Liu ◽  
Jing Wu ◽  
Lingai Pan ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Fungal Infections ◽  
Rna Virus ◽  
Paired Comparison ◽  
Final Analysis ◽  
Metagenomic Sequencing ◽  
Next Generation ◽  
Dna Virus ◽  
Virus Identification ◽  
Generation Sequencing

Objectives: To evaluate the performance of metagenomic next generation sequencing (mNGS) using adequate criteria for the detection of pathogens in lower respiratory tract (LRT) samples with a paired comparison to conventional microbiology tests (CMT).Methods: One hundred sixty-seven patients were reviewed from four different intensive care units (ICUs) in mainland China during 2018 with both mNGS and CMT results of LRT samples available. The reads per million ratio (RPMsample/RPMnon−template−control ratio) and standardized strictly mapped reads number (SDSMRN) were the two criteria chosen for identifying positive pathogens reported from mNGS. A McNemar test was used for a paired comparison analysis between mNGS and CMT.Results: One hundred forty-nine cases were counted into the final analysis. The RPMsample/RPMNTC ratio criterion performed better with a higher accuracy for bacteria, fungi, and virus than SDSMRN criterion [bacteria (RPMsample/RPMNTC ratio vs. SDSMRN), 65.1 vs. 55.7%; fungi, 75.8 vs. 71.1%; DNA virus, 86.3 vs. 74.5%; RNA virus, 90.9 vs. 81.8%]. The mNGS was also superior in bacteria detection only if an SDSMRN ≥3 was used as a positive criterion with a paired comparison to culture [SDSMRN positive, 92/149 (61.7%); culture positive, 54/149 (36.2%); p &lt; 0.001]; however, it was outperformed with significantly more fungi and DNA virus identification when choosing both criteria for positive outliers [fungi (RPMsample/RPMNTC ratio vs. SDSMRN vs. culture), 23.5 vs. 29.5 vs. 8.7%, p &lt; 0.001; DNA virus (RPMsample/RPMNTC ratio vs. SDSMRN vs. PCR), 14.1 vs. 20.8 vs. 11.8%, p &lt; 0.05].Conclusions: Metagenomic next generation sequencing may contribute to revealing the LRT infection etiology in hospitalized groups of potential fungal infections and in situations with less access to the multiplex PCR of LRT samples from the laboratory by choosing a wise criterion like the RPMsample/RPMNTC ratio.

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