Diagnosis of Haemophilus influenzae Pneumonia by Nanopore 16S Amplicon Sequencing of Sputum.
Ontology highlight
ABSTRACT:
SUBMITTER: Moon J
PROVIDER: S-EPMC6154133 | biostudies-literature | 2018 Oct
REPOSITORIES: biostudies-literature
Publications
Diagnosis of Haemophilus influenzae Pneumonia by Nanopore 16S Amplicon Sequencing of Sputum.
Moon Jangsup J Jang Yoonhyuk Y Kim Narae N Park Wan Beom WB Park Kyung-Il KI Lee Soon-Tae ST Jung Keun-Hwa KH Kim Manho M Lee Sang Kun SK Chu Kon K
Emerging infectious diseases 20181017 10
We used deep sequencing of the 16S rRNA gene from sputum to identify Haemophilus influenza in a patient with community-acquired pneumonia. This method may be more effective than conventional diagnostic tests in pneumonia patients because of its speed and sensitivity. ...[more]
Similar Datasets
Project description:Background16S rRNA amplicon sequencing is an accurate method of detecting microbial infection without culture. It is unclear if sequencing has additional benefits over routine diagnostic methods for Helicobacter pylori testing.MethodsWe enrolled Mongolian volunteers with dyspepsia. Using routine diagnostic methods, positive H. pylori was defined as positive results on histology/immunohistochemistry, culture, rapid urease test, or serology; negative H. pylori was defined by negative results from all these tests. We performed 16S rRNA sequencing on gastric biopsy specimens and calculated cutoffs for operational taxonomic units (OTUs) and relative abundance (RA) to define positive results using ROC curves.ResultsWe examined 161 individuals with a mean age of 43.6 years, and 64.6% were women. Using routine diagnostic methods, 122 (75.8%) participants were H. pylori positive, the sensitivity and specificity for 16S rRNA sequencing were 94.3% and 82.1% or 93.4% and 82.1% when cutoff values were set to 1113 (OTU number) or 4.4% RA, respectively (both p < .001). When combining the validated values, the concordance rate was high (91.1%); however, 16S rRNA sequencing had additional positive yield in 9 cases (5.6%) compared with routine diagnostic methods, and much greater additional positive yield compared to histopathology/IHC, culture, RUT, serology separately with 12 (7.4%), 37 (23.0%) and 43 (26.7%).Conclusion16S rRNA amplicon sequencing detects potentially important proportion of H. pylori-positive cases that test negative with routine diagnostic methods. The quantitative number of H. pylori can help to understand how it can be changing by diseases and RA give opportunity to understand how H. pylori communicate with other microbiota.
| S-EPMC8122063 | biostudies-literature
Project description:Current routine real-time PCR methods used for the point-of-care diagnosis of infectious meningitis do not allow for one-shot genotyping of the pathogen, as in the case of deadly Haemophilus influenzae meningitis. Real-time PCR diagnosed H. influenzae meningitis in a 22-year-old male patient, during his hospitalisation following a more than six-metre fall. Using an Oxford Nanopore Technologies real-time sequencing run in parallel to real-time PCR, we detected the H. influenzae genome directly from the cerebrospinal fluid sample in six hours. Furthermore, BLAST analysis of the sequence encoding for a partial DUF417 domain-containing protein diagnosed a non-b serotype, non-typeable H.influenzae belonging to lineage H. influenzae 22.1-21. The Oxford Nanopore metagenomic next-generation sequencing approach could be considered for the point-of-care diagnosis of infectious meningitis, by direct identification of pathogenic genomes and their genotypes/serotypes.
| S-EPMC8069228 | biostudies-literature
Project description:BackgroundThe study of microbial communities and their applications have been leveraged by advances in sequencing techniques and bioinformatics tools. The Oxford Nanopore Technologies long-read sequencing by nanopores provides a portable and cost-efficient platform for sequencing assays. While this opens the possibility of sequencing applications outside specialized environments and real-time analysis of data, complementing the existing efficient library preparation protocols with streamlined bioinformatic workflows is required.ResultsHere we present NanoRTax, a Nextflow pipeline for nanopore 16S rRNA gene amplicon data that features state-of-the-art taxonomic classification tools and real-time capability. The pipeline is paired with a web-based visual interface to enable user-friendly inspections of the experiment in progress. NanoRTax workflow and a simulated real-time analysis were used to validate the prediction of adult Intensive Care Unit patient mortality based on full-length 16S rRNA sequencing data from respiratory microbiome samples.ConclusionsThis constitutes a proof-of-concept simulation study of how real-time bioinformatic workflows could be used to shorten the turnaround times in critical care settings and provides an instrument for future research on early-response strategies for sepsis.
| S-EPMC9522874 | biostudies-literature
Project description:High-throughput sequencing is a robust tool used for identifying and tracking pathogen outbreaks. Whole-genome sequencing of hepatitis A virus (HAV) remains poor due to ultra-low viral loads, limitations of next-generation sequencing technology, and its high costs in clinical applications. This study evaluated multiplex polymerase chain reaction (PCR)-based nanopore sequencing to obtain whole-genome sequences of HAV. The HAV genomes were obtained directly from patient specimens for a rapid molecular diagnosis of viral genotypes. Serum and stool samples were collected from six patients with hepatitis A infection. Amplicon-based nanopore sequencing was performed from the clinical specimens to identify HAV genotypes by acquiring nearly complete-genome sequences. TaqMan-based quantitative PCR (qPCR) was conducted to detect and quantify multiple HAV genes. Singleplex-based nanopore sequencing demonstrated high genome coverage rates (90.4-99.5%) of HAV within 8 h, at viral RNA loads of 10 to 105 copies/μL. TaqMan qPCR showed multiplex quantification of HAV genes namely, VP0, VP3, and 3C. This study provides useful insights into rapid molecular diagnosis during hepatitis A outbreaks and may ultimately augment public health disease surveillance in the hospital and epidemiology field.
| S-EPMC10337952 | biostudies-literature
Project description:Small subunit (SSU) ribosomal RNA (rRNA) gene amplicon sequencing is a foundational method in microbial ecology. Currently, short-read platforms are commonly employed for high-throughput applications of SSU rRNA amplicon sequencing, but at the cost of poor taxonomic classification due to limited fragment lengths. The Oxford Nanopore Technologies (ONT) platform can sequence full-length SSU rRNA genes, but its lower raw-read accuracy has so-far limited accurate taxonomic classification and de novo feature generation. Here, we present a sequencing workflow, termed ssUMI, that combines unique molecular identifier (UMI)-based error correction with newer (R10.4+) ONT chemistry and sample barcoding to enable high throughput near full-length SSU rRNA (e.g. 16S rRNA) amplicon sequencing. The ssUMI workflow generated near full-length 16S rRNA consensus sequences with 99.99% mean accuracy using a minimum subread coverage of 3×, surpassing the accuracy of Illumina short reads. The consensus sequences generated with ssUMI were used to produce error-free de novo sequence features with no false positives with two microbial community standards. In contrast, Nanopore raw reads produced erroneous de novo sequence features, indicating that UMI-based error correction is currently necessary for high-accuracy microbial profiling with R10.4+ ONT sequencing chemistries. We showcase the cost-competitive scalability of the ssUMI workflow by sequencing 87 time-series wastewater samples and 27 human gut samples, obtaining quantitative ecological insights that were missed by short-read amplicon sequencing. ssUMI, therefore, enables accurate and low-cost full-length 16S rRNA amplicon sequencing on Nanopore, improving accessibility to high-resolution microbiome science.
| S-EPMC11462149 | biostudies-literature
Project description:BackgroundSevere asthma (SA) encompasses several clinical phenotypes with a heterogeneous airway microbiome. We determined the phenotypes associated with a low α-diversity microbiome.MethodsMetagenomic sequencing was performed on sputum samples from SA participants. A threshold of 2 standard deviations below the mean of α-diversity of mild-moderate asthma and healthy control subjects was used to define those with an abnormal abundance threshold as relative dominant species (RDS).FindingsFifty-one out of 97 SA samples were classified as RDSs with Haemophilus influenzae RDS being most common (n = 16), followed by Actinobacillus unclassified (n = 10), Veillonella unclassified (n = 9), Haemophilus aegyptius (n = 9), Streptococcus pseudopneumoniae (n = 7), Propionibacterium acnes (n = 5), Moraxella catarrhalis (n = 5) and Tropheryma whipplei (n = 5). Haemophilus influenzae RDS had the highest duration of disease, more exacerbations in previous year and greatest number on daily oral corticosteroids. Hierarchical clustering of RDSs revealed a C2 cluster (n = 9) of highest relative abundance of exclusively Haemophilus influenzae RDSs with longer duration of disease and higher sputum neutrophil counts associated with enrichment pathways of MAPK, NF-κB, TNF, mTOR and necroptosis, compared to the only other cluster, C1, which consisted of 7 Haemophilus influenzae RDSs out of 42. Sputum transcriptomics of C2 cluster compared to C1 RDSs revealed higher expression of neutrophil extracellular trap pathway (NETosis), IL6-transignalling signature and neutrophil activation.ConclusionWe describe a Haemophilus influenzae cluster of the highest relative abundance associated with neutrophilic inflammation and NETosis indicating a host response to the bacteria. This phenotype of severe asthma may respond to specific antibiotics.
| S-EPMC11347389 | biostudies-literature
Project description:Nontypeable Haemophilus influenzae (NTHi) is an opportunistic pathogen which causes a variety of respiratory infections. The objectives of the study were to determine its antimicrobial susceptibility, to characterize the β-lactam resistance, and to establish a genetic characterization of NTHi isolates. Ninety-five NTHi isolates were analyzed by pulsed field gel electrophoresis (PFGE) and multi locus sequence typing (MLST). Antimicrobial susceptibility was determined by microdilution, and the ftsI gene (encoding penicillin-binding protein 3, PBP3) was PCR amplified and sequenced. Thirty (31.6%) isolates were non-susceptible to ampicillin (MIC ≥ 2 mg/L), with 10 of them producing β-lactamase type TEM-1 as a resistance mechanism. After ftsI sequencing, 39 (41.1%) isolates showed amino acid substitutions in PBP3, with Asn526 → Lys being the most common (69.2%). Eighty-four patients were successfully treated with amoxicillin/clavulanic acid, ceftriaxone and levofloxacin. Eight patients died due either to aspiration or complication of their comorbidities. In conclusion, NTHi causing CAP in adults shows high genetic diversity and is associated with a high rate of reduced susceptibility to ampicillin due to alterations in PBP3. The analysis of treatment and outcomes demonstrated that NTHi strains with mutations in the ftsI gene could be successfully treated with ceftriaxone or fluoroquinolones.
| S-EPMC3862678 | biostudies-literature
Project description:BackgroundSpecies-level genetic characterization of complex bacterial communities has important clinical applications in both diagnosis and treatment. Amplicon sequencing of the 16S ribosomal RNA (rRNA) gene has proven to be a powerful strategy for the taxonomic classification of bacteria. This study aims to improve the method for full-length 16S rRNA gene analysis using the nanopore long-read sequencer MinION™. We compared it to the conventional short-read sequencing method in both a mock bacterial community and human fecal samples.ResultsWe modified our existing protocol for full-length 16S rRNA gene amplicon sequencing by MinION™. A new strategy for library construction with an optimized primer set overcame PCR-associated bias and enabled taxonomic classification across a broad range of bacterial species. We compared the performance of full-length and short-read 16S rRNA gene amplicon sequencing for the characterization of human gut microbiota with a complex bacterial composition. The relative abundance of dominant bacterial genera was highly similar between full-length and short-read sequencing. At the species level, MinION™ long-read sequencing had better resolution for discriminating between members of particular taxa such as Bifidobacterium, allowing an accurate representation of the sample bacterial composition.ConclusionsOur present microbiome study, comparing the discriminatory power of full-length and short-read sequencing, clearly illustrated the analytical advantage of sequencing the full-length 16S rRNA gene.
| S-EPMC7836573 | biostudies-literature
Project description:As of July 2022, more than 16,000 laboratory-confirmed monkeypox (MPX) cases have been reported worldwide. Until recently, MPX was a rare viral disease seldom detected outside Africa. MPX virus (MPXV) belongs to the Orthopoxvirus (OPV) genus and is a genetically close relative of the Variola virus (the causative agent of smallpox). Following the eradication of smallpox, there was a significant decrease in smallpox-related morbidity and the population's immunity to other OPV-related diseases such as MPX. In parallel, there was a need for differential diagnosis between the different OPVs' clinical manifestations and diseases with similar symptoms (i.e., chickenpox, herpes simplex). The current study aimed to provide a rapid genetic-based diagnostic tool for accurate and specific identification of MPXV and additional related vesicle-forming pathogens. We initially assembled a list of 14 relevant viral pathogens, causing infectious diseases associated with vesicles, prone to be misdiagnosed as MPX. Next, we developed an approach that we termed rapid amplicon nanopore sequencing (RANS). The RANS approach uses diagnostic regions that harbor high homology in their boundaries and internal diagnostic SNPs that, when sequenced, aid the discrimination of each pathogen within a group. During a multiplex PCR amplification, a dA tail and a 5'-phosphonate were simultaneously added, thus making the PCR product ligation ready for nanopore sequencing. Following rapid sequencing (a few minutes), the reads were compared to a reference database and the nearest strain was identified. We first tested our approach using samples of known viruses cultured in cell lines. All the samples were identified correctly and swiftly. Next, we examined a variety of clinical samples from the 2022 MPX outbreak. Our RANS approach identified correctly all the PCR-positive MPXV samples and mapped them to strains that were sequenced during the 2022 outbreak. For the subset of samples that were negative for MPXV by PCR, we obtained definite results, identifying other vesicle-forming viruses: Human herpesvirus 3, Human herpesvirus 2, and Molluscum contagiosum virus. This work was a proof-of-concept study, demonstrating the potential of the RANS approach for rapid and discriminatory identification of a panel of closely related pathogens. The simplicity and affordability of our approach makes it straightforward to implement in any genetics lab. Moreover, other differential diagnostics panels might benefit from the implementation of the RANS approach into their diagnostics pipelines.
| S-EPMC9416277 | biostudies-literature
Project description:The integration of long-read sequencing technology, such as nanopore sequencing technology [Oxford Nanopore Technologies (ONT)], into routine diagnostic laboratories has the potential to transform bacterial infection diagnostics and improve patient management. Analysis of amplicons from long-read sequencing of the 16S rRNA gene generates a comprehensive view of the microbial community within clinical samples, significantly enhancing sensitivity and capacity to analyse mixed bacterial populations compared to short read sequencing approaches. This study evaluates various ONT sequencing approaches and library preparation kits to establish a reliable testing and quality framework for clinical implementation. This study highlights the critical importance of using well-characterized reference materials in validating and revalidating long-read sequencing methods, leveraging a combination of standardized reference materials and clinical samples to navigate the evolving landscape of microbial diagnostics. It presents a robust validation framework for laboratory accreditation and outlines a methodology for comparing the performance of newer ONT chemistries with earlier versions. Additionally, the study details the methods and quality control measures necessary for achieving more accurate and efficient diagnoses of bacterial infections, ultimately reducing time to treatment and enhancing patient outcomes.
| S-EPMC11922894 | biostudies-literature