scholarly journals 2050. Plasma Next-Generation Sequencing for Pathogen Detection in Pediatric Patients at Risk for Invasive Fungal Infection

2018 ◽  
Vol 5 (suppl_1) ◽  
pp. S598-S598 ◽  
Author(s):  
Amy Armstrong ◽  
Jenna Rossoff ◽  
Romielle Aquino ◽  
Desiree Hollemon ◽  
David Hong ◽  
...  
Keyword(s):  
At Risk ◽  
Next Generation Sequencing ◽  
Fungal Infection ◽  
Invasive Fungal Infection ◽  
Pathogen Detection ◽  
Pediatric Patients ◽  
Next Generation ◽  
Patients At Risk ◽  
Generation Sequencing

scholarly journals 265. Clinical Epidemiology of Invasive Fungal Infection with Aspergillus and Mucor Species in a Tertiary Children’s Hospital

2019 ◽  
Vol 6 (Supplement_2) ◽  
pp. S147-S147
Author(s):  
Zacharoula Oikonomopoulou ◽  
Sameer Patel ◽  
Jacquie Toia ◽  
William Muller
Keyword(s):  
Next Generation Sequencing ◽  
Fungal Infection ◽  
Invasive Fungal Infection ◽  
Small Sample ◽  
Fungal Culture ◽  
Next Generation ◽  
Hematopoietic Stem ◽  
Children's Hospital ◽  
Children’S Hospital ◽  
Generation Sequencing

Abstract Background Patients undergoing hematopoietic stem cell transplantation and patients with hematologic malignancies are at increased risk for acquiring invasive fungal infection (IFI) due to immune system impairment from chemotherapy. Affected patients require prolonged antifungal therapy with the risk of associated toxicity and extended hospitalization due to delay of accurate diagnosis. There is a lack of effective serologic biomarkers and hesitancy to proceed with tissue diagnosis due to thrombocytopenia or other associated risks. Mortality in oncology patients with invasive mycoses is high, with pediatric mortality rates of 30–40% at 12 weeks following diagnosis. Methods All patients that were admitted to Lurie Children’s Hospital between January 2014 and December 2018 and received voriconazole, ambisome, posaconazole and isavuconazole were identified. The following data were retrospectively collected: CT chest and sinus, (1,3)-β-d-Glucan and Aspergillus galactomannan, ANC and ALC at diagnosis, blood next-generation sequencing, tissue 18s rRNA, fungal culture, duration of neutropenia and lymphopenia, site of infection, time between underlying diagnosis and development of IFI, surgical intervention and associated mortality. Results A total of 94 unique patients that received voriconazole were identified. There were 8 proven cases of invasive Aspergillus infection the past 5 years, 50% male, mean age 14 years. Only 25% of patients had positive serum Aspergillus galactomannan and 37.5% had positive β-d-Glucan. Seven cases were due to Aspergillus fumigatus and one case was due to Aspergillus flavus. There were 9 patients with mucormycosis and all but one were culture positive. Three patients with Mucor had mold identification in blood next-generation sequencing prior to surgery. Mucor associated mortality was 22.2%. Conclusion The majority of pediatric patients with invasive aspergillosis did not have characteristic chest CT imaging findings and serum Aspergillus galactomannan was usually negative.The was no associated mortality in invasive Aspergillus cases, whereas the mortality rate of invasive mucormycosis was 22.2%. Although we have a small sample size, this is significantly lower compared with published literature. Disclosures All authors: No reported disclosures.

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.

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 Genotypic Characteristics of Hepatoblastoma as Detected by Next Generation Sequencing and Their Correlation With Clinical Efficacy

2021 ◽  
Vol 11 ◽  
Author(s):  
Huimin Hu ◽  
Weiling Zhang ◽  
Tian Zhi ◽  
Jing Li ◽  
Yuan Wen ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Survival Rate ◽  
Mismatch Repair ◽  
Pediatric Patients ◽  
Next Generation ◽  
Expression Levels ◽  
Dna Mismatch ◽  
Mismatch Repair Status ◽  
Generation Sequencing ◽  
Potential Biomarkers

BackgroundHepatoblastoma (HB) is the most common malignant embryonic liver tumor type in children under 3 years of age. In the present study, the next generation sequencing (NGS) method was used to detect the genotype characteristics of HB and summarize the correlation between the common mutation genotypes noted in this disease and the clinical treatment and prognosis. The results may aid clinical prognosis and the successful application of targeted drugs.MethodsInitially, DNA was extracted from tumor tissue specimens and peripheral blood derived from 19 pediatric patients with HB. Subsequently, DNA panel and NGS methods were used to detect tumor diagnosis and the expression levels of treatment-associated genes, followed by the summary of genotype characteristics. In addition, in order to further assess the application of immunotherapy in HB, immunohistochemical detection of programmed cell death 1 ligand 1 (PDL1) was performed in combination with tumor mutation burden (TMB) and DNA mismatch repair status analysis. Furthermore, the clinical treatment effect and prognosis of the pediatric patients were statistically analyzed according to the characteristics of the genotype. Overall prognosis and prognostic analyses in different groups were performed by Kaplan-Meier and log-rank tests, respectively. Finally, expression validation and diagnostic analysis of commonly reported genes were performed in the GSE75271 dataset, which was obtained from the Gene Expression Omnibus (GEO) database.ResultsIn the present study, certain mutated genes, including nuclear factor erythroid 2-related factor 2 (NFE2L2), catenin β1 (CTNNB1), MYCN, tumor protein p53, axis inhibition protein 1 (AXIN1) and adenomatous polyposis coli (APC) were associated with the pathogenesis of HB. During TMB and DNA mismatch repair status analyses, pediatric patients had a low TMB. All of them did not present with microsatellite instability. The immunohistochemical results indicated lower expression levels of PDL1 in HB. The complete remission (CR) rate of pediatric patients in the gene abnormality group was lower than that of the non-reported disease-associated gene abnormality group. The 2-year overall survival rate and disease-free survival rate of 19 pediatric patients with HB were 72.1% and 42.4%, respectively. Receiver operating characteristic (ROC) analysis demonstrated that CTNNB1, NFE2L2, AXIN1, APC, MYCN and insulin growth factor 2 (IGF2) may be potential biomarkers that could be used for the diagnosis of HB.ConclusionThe genotype changes in HB were more common and the CR rate of the pediatric patients with an altered genotype was lower than that of pediatric patients without an altered genotype. In addition, pediatric patients with HB exhibited lower TMB compared with adult patients. Moreover, the data indicated that CTNNB1, NFE2L2, AXIN1, APC, MYCN and IGF2 may be potential biomarkers that can be used for the diagnosis of HB.

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