Use of Metagenomic Next-Generation Sequencing to Identify Pathogens in Pediatric Osteoarticular Infections

Abstract Background Osteoarticular infections (OAI) are frequently encountered in children. Treatment may be guided by isolation of a pathogen; however, operative cultures are often negative. Metagenomic next-generation sequencing (mNGS) allows for broad and sensitive pathogen detection that is culture-independent. We sought to evaluate the diagnostic utility of mNGS in comparison to culture and usual care testing to detect pathogens in acute osteomyelitis and/or septic arthritis in children. Methods This was a single-site study to evaluate the use of mNGS in comparison to culture to detect pathogens in acute pediatric osteomyelitis and/or septic arthritis. Subjects admitted to a tertiary children’s hospital with suspected OAI were eligible for enrollment. We excluded subjects with bone or joint surgery within 30 days of admission or with chronic osteomyelitis. Operative samples were obtained at the surgeon’s discretion per standard care (fluid or tissue) and based on imagining and operative findings. We compared mNGS to culture and usual care testing (culture and PCR) from the same site. Results We recruited 42 subjects over the enrollment period. mNGS of the operative samples identified a pathogen in 26 subjects compared to 19 subjects in whom culture identified a pathogen. In four subjects, mNGS identified a pathogen where combined usual care testing (culture and PCR) was negative. Positive predictive agreement and negative predictive agreement both were 93.0% for mNGS. Conclusion In this single site prospective study of pediatric OAI, we demonstrated the diagnostic utility of mNGS testing in comparison to culture and usual care (culture and PCR) from operative specimens.

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329. Performance of Next Generation Sequencing in Isolating a Pathogen in Pediatric Osteoarticular Infections

Open Forum Infectious Diseases ◽

10.1093/ofid/ofaa439.525 ◽

2020 ◽

Vol 7 (Supplement_1) ◽

pp. S236-S237

Author(s):

Nanda Ramchandar ◽

Jessica Burns ◽

Andrew Pennock ◽

Christopher R Cannavino ◽

Lauge Farnaes

Keyword(s):

Next Generation Sequencing ◽

Septic Arthritis ◽

Standard Care ◽

Pediatric Population ◽

Single Site ◽

Acute Osteomyelitis ◽

Next Generation ◽

Pathogenic Organism ◽

Osteoarticular Infections ◽

Generation Sequencing

Abstract Background Osteoarticular infections are often encountered in the pediatric population. Therapy is guided by isolation of a putative organism, however, operative cultures are often negative. Next generation sequencing (NGS) allows for more sensitive sampling of body compartments generally considered sterile. We sought to evaluate the utility of NGS in comparison to culture in detecting a pathogenic organism in acute osteomyelitis and septic arthritis in children. Methods This was a single-site study to evaluate the utility of NGS in comparison to culture in detecting a pathogenic organism in acute osteomyelitis and septic arthritis in children. Eligible patients were all patients with osteomyelitis or septic arthritis admitted to Rady Children’s Hospital from July 2019 through July 2020. We excluded any patients with bone or joint surgery within 30 days prior to admission. Operative samples were chosen at the surgeon’s discretion (joint aspirate, synovium, or bone) based on operative findings. We compared NGS testing to standard care culture from the same site. Results We enrolled 41 subjects. NGS of the operative samples identified a pathogen in 26 (63.4%) patients versus 18 (43.9%) by culture. Operative culture missed the diagnosis in 10 cases, though PCR identified the organism in 6 of those cases (5 were cases in which Kingella kingae was identified). In 4 subjects, NGS identified a putative organism where standard care testing (either PCR or culture) was negative. NGS was falsely positive in 1 subject and falsely negative for one other subject. Sensitivity was 96.3% (CI 95%, 81.0–99.9%) and Specificity was 92.9% (CI 95%, 66.1–99.8) for NGS versus 64.3% (CI 95%, 44.1–81.4) and 84.6% (CI 95%, 54.6–99.9%) for culture respectively. Conclusion In this single site prospective study of pediatric osteoarticular infections, we demonstrate improved sensitivity and specificity of NGS testing when compared to standard culture. Disclosures All Authors: No reported disclosures

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Diagnostic utility of next-generation sequencing panel tests in the diagnosis of skeletal dysplasias

Molecular Genetics and Metabolism ◽

10.1016/s1096-7192(21)00428-5 ◽

2021 ◽

Vol 132 ◽

pp. S221-S222

Author(s):

Alicia Scocchia ◽

Tiia Kangas-Kontio ◽

Liisa Pelttari ◽

Kim Gall ◽

Inka Saarinen ◽

...

Keyword(s):

Next Generation Sequencing ◽

Diagnostic Utility ◽

Next Generation ◽

Skeletal Dysplasias ◽

Generation Sequencing

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Metagenomic Next-Generation Sequencing for Pathogen Detection and Transcriptomic Analysis in Pediatric Central Nervous System Infections

Open Forum Infectious Diseases ◽

10.1093/ofid/ofab104 ◽

2021 ◽

Author(s):

Nanda Ramchandar ◽

Nicole G Coufal ◽

Anna S Warden ◽

Benjamin Briggs ◽

Toni Schwarz ◽

...

Keyword(s):

Central Nervous System ◽

Nervous System ◽

Next Generation Sequencing ◽

Usual Care ◽

Transcriptomic Analysis ◽

Cns Infection ◽

Metagenomic Sequencing ◽

Next Generation ◽

Cns Infections ◽

Generation Sequencing

Abstract Background Pediatric central nervous system (CNS) infections are potentially life-threatening and may incur significant morbidity. Identifying a pathogen is important, both in terms of guiding therapeutic management, but also in characterizing prognosis. Usual care testing by culture and PCR is often unable to identify a pathogen. We examined the systematic application of metagenomic next-generation sequencing (mNGS) for detecting organisms and transcriptomic analysis of cerebrospinal fluid (CSF) in children with CNS infections. Methods We conducted a prospective multi-site study that aimed to enroll all children with a CSF pleocytosis and suspected CNS infection admitted to one of three tertiary pediatric hospitals during the study timeframe. After usual care testing had been performed, the remaining CSF was sent for mNGS and transcriptomic analysis. Results We screened 221 and enrolled 70 subjects over a 12-month recruitment period. A putative organism was isolated from CSF in 25 (35.7%) subjects by any diagnostic modality. mNGS of the CSF samples identified a pathogen in 20 (28.6%) subjects, which were also all identified by usual care testing. The median time to result was 38 hours. Conclusion Metagenomic sequencing of CSF has the potential to rapidly identify pathogens in children with CNS infections.

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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

Frontiers in Microbiology ◽

10.3389/fmicb.2021.641202 ◽

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.

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

Journal of Clinical Microbiology ◽

10.1128/jcm.03050-15 ◽

2016 ◽

Vol 54 (4) ◽

pp. 919-927 ◽

Cited By ~ 66

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.

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The Perils of Single‐Site Genetic Testing for Hereditary Cancer Syndromes in the Era of Next‐Generation Sequencing

The Oncologist ◽

10.1634/theoncologist.2017-0372 ◽

2018 ◽

Vol 23 (4) ◽

pp. 393-396

Author(s):

Nicole Casasanta ◽

Elizabeth Stark ◽

Allison McHenry ◽

Tara Biagi ◽

Rebecca Kaltman

Keyword(s):

Genetic Testing ◽

Next Generation Sequencing ◽

Hereditary Cancer ◽

Single Site ◽

Next Generation ◽

Hereditary Cancer Syndromes ◽

Cancer Syndromes ◽

Generation Sequencing

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Detection of Respiratory Pathogens in Parapneumonic Effusions by Hypothesis-free, Next-Generation Sequencing (NGS)

Open Forum Infectious Diseases ◽

10.1093/ofid/ofx162.044 ◽

2017 ◽

Vol 4 (suppl_1) ◽

pp. S17-S17

Author(s):

Krow Ampofo ◽

Andrew Pavia ◽

Anne J Blaschke ◽

Robert Schlaberg

Keyword(s):

Next Generation Sequencing ◽

Pathogen Detection ◽

Respiratory Pathogens ◽

Bacterial Identification ◽

Next Generation ◽

University Of Utah ◽

Species Specific ◽

Next Generation Sequencing Ngs ◽

Ngs Data ◽

Generation Sequencing

Abstract Background Species-specific polymerase chain reaction (PCR) testing of pleural fluid (PF) from children with parapneumonic effusion (PPE) has increased pathogen identification in pediatric PPE. However, a pathogen is not detected in 25–35% of cases. Hypothesis-free, next-generation sequencing (NGS) provides a more comprehensive alternative and has led to pathogen detection in PCR-negative samples. However, the utility of NGS in the evaluation of PF from children with PPE is unknown. Methods Archived PF (n = 20) from children younger than 18 years with PPE and hospitalized at Primary Children’s Hospital, Utah, in 2015 and previously tested by PCR were evaluated. Ten PCR-negative and 10 PCR-positive PF specimens were tested using RNA-seq at an average depth of 7.7×106 sequencing reads per sample. NGS data were analyzed with Taxonomer. We compared pathogens detected by blood and PF culture, PCR, and NGS. Results Overall, compared with blood/PF culture, PF PCR and PF NGS testing of PF increased bacterial identification from 15% to 50% (P < 0.05) and 65% (P = 0.003), respectively. Pathogen detection in PF by PCR and NGS were comparable (50 vs. 65%, p = NS) (Table). However, compared with PF PCR, NGS significantly increased detection of S. pyogenes (20% vs. 55%; P < 0.05), with 100% concordance when detected by PCR and culture. Detection of Fusobacterium spp. (10 vs. 10%) by PF NGS and PF PCR were comparable. In contrast, there was no detection of S. pneumoniae (15 vs. 0%) by PF NGS compared with PF PCR. Conclusion PF NGS testing significantly improves bacterial identification and comparable to PF PCR testing, which can help inform antimicrobial selection. However there were differences in detection of S. pneumoniae and S. pyogenes. Further studies of NGS testing of PF of children with PPE are needed to assess its potential in the evaluation of PPE in children. Disclosures A. J. Blaschke, BioFire Diagnostics LLC: Collaborator, Have intellectual property in BioFire Diagnostics through the University of Utah and Investigator, Licensing agreement or royalty and Research support; R. Schlaberg, IDbyDNA: Co-founder, Consultant and Shareholder, Stock

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