Diagnosis of Capnocytophaga canimorsus Sepsis by Whole-Genome Next-Generation Sequencing.
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ABSTRACT:
SUBMITTER: Abril MK
PROVIDER: S-EPMC5047422 | biostudies-literature | 2016 Sep
REPOSITORIES: biostudies-literature
Publications
Diagnosis of <i>Capnocytophaga canimorsus</i> Sepsis by Whole-Genome Next-Generation Sequencing.
Abril Maria K MK Barnett Adam S AS Wegermann Kara K Fountain Eric E Strand Andrew A Heyman Benjamin M BM Blough Britton A BA Swaminathan Aparna C AC Sharma-Kuinkel Batu B Ruffin Felicia F Alexander Barbara D BD McCall Chad M CM Costa Sylvia F SF Arcasoy Murat O MO Hong David K DK Blauwkamp Timothy A TA Kertesz Michael M Fowler Vance G VG Kraft Bryan D BD
Open forum infectious diseases 20160712 3
We report the case of a 60-year-old man with septic shock due to <i>Capnocytophaga canimorsus</i> that was diagnosed in 24 hours by a novel whole-genome next-generation sequencing assay. This technology shows great promise in identifying fastidious pathogens, and, if validated, it has profound implications for infectious disease diagnosis. ...[more]
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Project description:Capnocytophaga canimorsus is a gram-negative rod that can be transmitted primarily by dog bites. This life-threatening organism commonly causes sepsis in patients with splenectomy or alcoholism. A 53-year-old rheumatoid arthritis male treated with methotrexate (MTX) for 5 years was admitted for a 4-day history of fever and dyspnea. He had been bitten on a finger by the family dog 4 days before onset. Laboratory tests revealed pancytopenia, acute renal failure, and evidence of disseminated intravascular coagulation, and he subsequently developed acute respiratory distress syndrome. Furthermore, blood cultures grew gram-negative bacilli and despite intensive treatment, he died 5 days after admission. Later, C. canimorsus was identified from his culture samples using a species-specific polymerase chain reaction. C. canimorsus infections should be considered in the differential diagnosis of sepsis for immunocompromised hosts following animal bites.
| S-EPMC5554928 | biostudies-other
Project description:Capnocytophaga canimorsus, a commensal bacterium from canine oral flora, has been isolated throughout the world from severe human infections caused by dog bites. Due to the low level of evolutionary relationship to Proteobacteria, genetic methods suitable for the genus Capnocytophaga needed to be established. Here, we show that Tn4351, derived from Bacteroides fragilis, could be introduced by conjugation into C. canimorsus and conferred resistance to erythromycin. By mapping and sequencing a naturally occurring plasmid isolated from a clinical isolate of C. canimorsus, we identified a repA gene that allowed us to construct Escherichia coli-Capnocytophaga shuttle vectors. Most commonly used antibiotic markers were not functional in C. canimorsus, but cefoxitin (cfxA), tetracycline (tetQ), and erythromycin (ermF) resistances could be used as markers for plasmid maintenance in C. canimorsus and even in some other Capnocytophaga spp. Shuttle vectors were introduced into C. canimorsus either by conjugation using the origin of transfer (oriT) of RP4 or by electrotransformation. Taking advantage of the promoter of ermF, an expression vector was constructed. Finally, a method that allows site-directed mutagenesis is described. All these genetic tools pave the way, not only for molecular studies of the pathogenesis of C. canimorsus, but also for studies of other oral Capnocytophaga species.
| S-EPMC2570293 | biostudies-literature
Project description:BackgroundRapid diagnosis and appropriate treatment is imperative in bacterial sepsis due increasing risk of mortality with every hour without appropriate antibiotic therapy. Atypical infections with fastidious organisms may take more than 4 days to diagnose leading to calls for improved methods for rapidly diagnosing sepsis. Capnocytophaga canimorsus is a slow-growing, fastidious gram-negative bacillus which is a common commensal within the mouths of dogs, but rarely cause infections in humans. C. canimorsus sepsis risk factors include immunosuppression, alcoholism and elderly age. Here we report on the application of emerging nanopore sequencing methods to rapidly diagnose an atypical case of C. canimorsus septic shock.Case presentationA 62 year-old female patient was admitted to an intensive care unit with septic shock and multi-organ failure six days after a reported dog bite. Blood cultures were unable to detect a pathogen after 3 days despite observed intracellular bacilli on blood smears. Real-time nanopore sequencing was subsequently employed on whole blood to detect Capnocytophaga canimorsus in 19 h. The patient was not immunocompromised and did not have any other known risk factors. Whole-genome sequencing of clinical sample and of the offending dog's oral swabs showed near-identical C. canimorsus genomes. The patient responded to antibiotic treatment and was discharged from hospital 31 days after admission.ConclusionsUse of real-time nanopore sequencing reduced the time-to-diagnosis of Capnocytophaga canimorsus in this case from 6.25 days to 19 h. Capnocytophaga canimorsus should be considered in cases of suspected sepsis involving cat or dog contact, irrespective of the patient's known risk factors.
| S-EPMC6657077 | biostudies-literature
Project description:Accurate molecular diagnosis of monogenic non-autoimmune neonatal diabetes mellitus (NDM) is critical for patient care, as patients carrying a mutation in KCNJ11 or ABCC8 can be treated by oral sulfonylurea drugs instead of insulin therapy. This diagnosis is currently based on Sanger sequencing of at least 42 PCR fragments from the KCNJ11, ABCC8, and INS genes. Here, we assessed the feasibility of using the next-generation whole exome sequencing (WES) for the NDM molecular diagnosis.We carried out WES for a patient presenting with permanent NDM, for whom mutations in KCNJ11, ABCC8 and INS and abnormalities in chromosome 6q24 had been previously excluded. A solution hybridization selection was performed to generate WES in 76 bp paired-end reads, by using two channels of the sequencing instrument. WES quality was assessed using a high-resolution oligonucleotide whole-genome genotyping array. From our WES with high-quality reads, we identified a novel non-synonymous mutation in ABCC8 (c.1455G>C/p.Q485H), despite a previous negative sequencing of this gene. This mutation, confirmed by Sanger sequencing, was not present in 348 controls and in the patient's mother, father and young brother, all of whom are normoglycemic.WES identified a novel de novo ABCC8 mutation in a NDM patient. Compared to the current Sanger protocol, WES is a comprehensive, cost-efficient and rapid method to identify mutations in NDM patients. We suggest WES as a near future tool of choice for further molecular diagnosis of NDM cases, negative for chr6q24, KCNJ11 and INS abnormalities.
| S-EPMC2964316 | biostudies-literature
Project description:Capnocytophaga canimorsus is a commensal Gram-negative bacterium, originally isolated from a dog's mouth, that causes septicemia in humans. C. canimorsus has the unusual ability to feed on host cells, including phagocytes. This capacity depends on surface-exposed glycan-foraging systems. Here we present the first complete genome sequence of a C. canimorsus strain (Cc5).
| S-EPMC3187460 | biostudies-literature
Project description:Capnocytophaga canimorsus is a dog oral commensal that causes rare but severe infections in humans. C. canimorsus was recently shown to be endowed with a capsular polysaccharide implicated in resistance to the innate immune system of the host. Here, we developed the first C. canimorsus capsular serotyping scheme. We describe nine different serovars (A to I), and this serotyping scheme allowed typing of 25/25 isolates from human infections but only 18/52 isolates from dog mouths, indicating that the repertoire of capsules in the species is vast. However, while only three serovars (A, B, and C) covered 88% of the human isolates tested (22/25), they covered only 7.7% of the dog isolates (4/52). Serovars A, B, and C were found 22.9-, 14.6-, and 4.2-fold more often, respectively, among human isolates than among dog isolates, with no geographical bias, implying that isolates endowed with these three capsular types are more virulent for humans than other isolates. Capsular serotyping would thus allow identification of virulent isolates in dogs, which could contribute to the prevention of these infections. To this end, we developed a PCR typing method based on the amplification of specific capsular genes.
| S-EPMC5442547 | biostudies-literature
Project description:BackgroundTo evaluate the value of metagenomic next-generation sequencing (mNGS) for the early diagnosis of psittacosis, and to investigate its epidemiology by whole-genome capture.MethodsTwenty-one bronchoalveolar lavage fluid (BALF) and blood samples of 16 psittacosis patients from multiple centers during August 2019 to September 2021 were analyzed retrospectively. mNGS with normal datasets (10 M 75-bp single-end reads after sequencing) and larger datasets (30 M 150-bp paired-end reads after sequencing) as well as quantitative real-time polymerase chain reaction (qPCR) were used to detect the pathogen. Also, whole-genome capture of Chlamydophila psittaci was applied to draw the phylogenetic tree.ResultsmNGS successfully detected the pathogen in all 16 cases (100%), while qPCR was positive only in 5 out of 10 cases (50%), indicating a significantly higher sensitivity of mNGS than qPCR (p < 0.01). BALF-mNGS performed better than blood-mNGS (16/16 versus 3/5, p < 0.05). In addition, larger datasets (the read counts have tripled, and the base number was 12-fold larger compared to clinical mNGS with a normal dataset) of mNGS showed significantly increased contents of human DNA (p < 0.05) and decreased reads per million of the pathogen, suggesting no improvement. Whole-genome capture results of five samples (>60% coverage and >1 depth) were used to construct the phylogenetic tree.ConclusionSignificant advantages of mNGS with normal datasets were demonstrated in early diagnosing psittacosis. It is the first study to use whole-genome capture to analyze C. psittaci epidemiological information.
| S-EPMC9207344 | biostudies-literature
Project description:IntroductionSepsis and septic shock are the most severe forms of infection affecting predominantly elderly people, preterm and term neonates, and young infants. Even in high-income countries sepsis causes about 8% of admissions to pediatric intensive care units (PICUs). Early diagnosis, rapid anti-infective treatment, and prompt hemodynamic stabilization are crucial for patient survival. In this context, it is essential to identify the causative pathogen as soon as possible to optimize antimicrobial treatment. To date, culture-based diagnostic procedures (e.g., blood cultures) represent the standard of care. However, they have 2 major problems: on the one hand, in the case of very small sample volumes (and thus usually in children), they are not sufficiently sensitive. On the other hand, with a time-to-result of 2 to 5 days, blood cultures need a relatively long time for the anti-infective therapy to be calculated. To overcome these problems, culture-independent molecular diagnostic procedures such as unbiased sequence analysis of circulating cell-free DNA (cfDNA) from plasma samples of septic patients by next-generation sequencing (NGS) have been tested successfully in adult septic patients. However, these results still need to be transferred to the pediatric setting.MethodsThe Next GeneSiPS-Trial is a prospective, observational, non-interventional, multicenter study used to assess the diagnostic performance of an NGS-based approach for the identification of causative pathogens in (preterm and term) neonates (d1-d28, n = 50), infants (d29 to <1 yr, n = 50), and toddlers (1 yr to <5 yr, n = 50) with suspected or proven severe sepsis or septic shock (according to the pediatric sepsis definition) by the use of the quantitative sepsis indicating quantifier (SIQ) score in comparison to standard of care (culture-based) microbiological diagnostics. Potential changes in anti-infective treatment regimens based on these NGS results will be estimated retrospectively by a panel of 3 independent clinical specialists.DiscussionNeonates, infants, and young children are significantly affected by sepsis. Fast and more sensitive diagnostic approaches are urgently needed. This prospective, observational, non-interventional, multicenter study seeks to evaluate an NGS-based approach in critically ill children suffering from sepsis.Trial registrationDRKS-ID: DRKS00015705 (registered October 24, 2018). https://www.drks.de/drks_web/navigate.do?navigationId=trial.HTML&TRIAL_ID=DRKS00015705.
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Project description:Telomeres are the ends of eukaryotic chromosomes, consisting of consecutive short repeats that protect chromosome ends from degradation. Telomeres shorten with each cell division, leading to replicative cell senescence. Deregulation of telomere length homeostasis is associated with the development of various age-related diseases and cancers. A number of experimental techniques exist for telomere length measurement; however, until recently, the absence of tools for extracting telomere lengths from high-throughput sequencing data has significantly obscured the association of telomere length with molecular processes in normal and diseased conditions. We have developed Computel, a program in R for computing mean telomere length from whole-genome next-generation sequencing data. Computel is open source, and is freely available at https://github.com/lilit-nersisyan/computel. It utilizes a short-read alignment-based approach and integrates various popular tools for sequencing data analysis. We validated it with synthetic and experimental data, and compared its performance with the previously available software. The results have shown that Computel outperforms existing software in accuracy, independence of results from sequencing conditions, stability against inherent sequencing errors, and better ability to distinguish pure telomeric sequences from interstitial telomeric repeats. By providing a highly reliable methodology for determining telomere lengths from whole-genome sequencing data, Computel should help to elucidate the role of telomeres in cellular health and disease.
| S-EPMC4414351 | biostudies-literature
Project description:NGPS is a method for de-novo, full-length protein sequencing in high throughput. The method is based on cleavage of the protein at semi-random sites by microwave-assisted acid hydrolysis (MAAH), enrichment of LC-MS/MS amenable peptides from the hydrolysate by solid-phase-extraction, LC-MS/MS analysis, de-novo long peptide tag sequencing of resulting peptides and assembly of peptide tags into consensus contigs.
2017-04-03 | PXD003804 | Pride