scholarly journals Rapid Identification of Pathogens Associated With Ventilator-Associated Pneumonia By Nanopore Sequencing

2021 ◽  
Author(s):  
Nan Wu ◽  
Piyush Ranjan ◽  
Changyu Tao ◽  
Chao Liu ◽  
Ence Yang ◽  
...  
Keyword(s):  
Pathogenic Bacteria ◽  
Rapid Identification ◽  
Construction Method ◽  
Ventilator Associated Pneumonia ◽  
Metagenomic Sequencing ◽  
Average Sensitivity ◽  
Local Data ◽  
Library Construction ◽  
Peking University ◽  
Pcr Method

Abstract BACKGROUND: Etiology detection is crucial in diagnose and treatment of ventilator associated pneumonia (VAP). However, the detection method is in need of improvement. In this study, we used Nanopore sequence to build a quick detection protocol and compared the efficiency of different methods on 7 common VAP pathogens.METHODS: Endotracheal aspirate (ETA) of 83 patients with suspected VAP from Peking University Third Hospital were collected, saponin were used to deplete host genomes, and PCR or non-PCR library construction method were used and compared for sequencing. MinION and local data analysis methods were used for sequencing and data analysis.RESULTS: Saponin depletion effectively removed 11 of 12 human genomes, while most pathogenic bacteria genome results showed no statistical difference except for S.pneumoniae who had a 0.31 times depletion. Meanwhile, the average sequence time reduced from 19.2hrs to 1.73hrs. Compared with PCR library construction method, the non-PCR method has a better average sensitivity (84.17% VS 81.35%) and specificity (97.42% VS 88.52%), while the non-PCR costs less time. The whole method takes 5-6hrs from ETA extraction to pathogen classification. After analyzed the 7 pathogens enrolled, the average sensitivity of metagenomic sequencing were about 2.4 times higher than that of clinical culture (91.79%VS 37.92%), and the average specificity was 98.08%.CONCLUSIONS: Using saponin to remove human genome and non-PCR method to build library can be used for identification of pathogens in ETA of VAP patients within 6 hours by MinION, which provides a new approach for rapid identification of pathogens in clinical departments.

scholarly journals Rapid identification of pathogens associated with ventilator-associated pneumonia by Nanopore sequencing

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Nan Wu ◽  
Piyush Ranjan ◽  
Changyu Tao ◽  
Chao Liu ◽  
Ence Yang ◽  
...  
Keyword(s):  
Data Analysis ◽  
Bacterial Genome ◽  
Pcr Amplification ◽  
Rapid Identification ◽  
Ventilator Associated Pneumonia ◽  
Metagenomic Sequencing ◽  
Nanopore Sequencing ◽  
Average Sensitivity ◽  
Amplification Method ◽  
Significant Difference

Abstract Background Aetiology detection is crucial in the diagnosis and treatment of ventilator-associated pneumonia (VAP). However, the detection method needs improvement. In this study, we used Nanopore sequencing to build a quick detection protocol and compared the efficiency of different methods for detecting 7 VAP pathogens. Methods The endotracheal aspirate (ETA) of 83 patients with suspected VAP from Peking University Third Hospital (PUTH) was collected, saponins were used to deplete host genomes, and PCR- or non-PCR-amplified library construction methods were used and compared. Sequence was performed with MinION equipment and local data analysis methods were used for sequencing and data analysis. Results Saponin depletion effectively removed 11 of 12 human genomes, while most pathogenic bacterial genome results showed no significant difference except for S. pneumoniae. Moreover, the average sequence time decreased from 19.6 h to 3.62 h. The non-PCR amplification method and PCR amplification method for library build has a similar average sensitivity (85.8% vs. 86.35%), but the non-PCR amplification method has a better average specificity (100% VS 91.15%), and required less time. The whole method takes 5–6 h from ETA extraction to pathogen classification. After analysing the 7 pathogens enrolled in our study, the average sensitivity of metagenomic sequencing was approximately 2.4 times higher than that of clinical culture (89.15% vs. 37.77%), and the average specificity was 98.8%. Conclusions Using saponins to remove the human genome and a non-PCR amplification method to build libraries can be used for the identification of pathogens in the ETA of VAP patients within 6 h by MinION, which provides a new approach for the rapid identification of pathogens in clinical departments.

PCR Detection of Alternaria spp. in Processed Foods, Based on the Internal Transcribed Spacer Genetic Marker

2011 ◽  
Vol 74 (2) ◽  
pp. 240-247 ◽  
Author(s):  
MIGUELÁNGEL PAVÓN ◽  
ISABEL GONZÁLEZ ◽  
MARÍA ROJAS ◽  
NICOLETTE PEGELS ◽  
ROSARIO MARTÍN ◽  
...  
Keyword(s):  
Food Processing ◽  
Internal Transcribed Spacer ◽  
Rapid Identification ◽  
Food Samples ◽  
Pcr Analysis ◽  
Infant Food ◽  
Rrna Gene ◽  
Tomato Pulp ◽  
Pcr Method ◽  
Fungal Contaminants

The genus Alternaria is considered one of the most important fungal contaminants of vegetables, fruits, and cereals, producing several mycotoxins that can withstand food processing methods. Conventional methods for Alternaria identification and enumeration are laborious and time-consuming, and they might not detect toxigenic molds inactivated by food processing. In this study, a PCR method has been developed for the rapid identification of Alternaria spp. DNA in foodstuffs, based on oligonucleotide primers targeting the internal transcribed spacer (ITS) 1 and ITS2 regions of the rRNA gene. The specificity of the Alternaria-specific primer pair designed (Dir1ITSAlt–Inv1ITSAlt) was verified by PCR analysis of DNA from various Alternaria spp., and also from several fungal, bacterial, yeast, animal, and plant species. The detection limit of the method was 102 CFU/ml in viable culture, heated culture, or experimentally inoculated tomato pulp. The applicability of the method for detection of Alternaria spp. DNA in foodstuffs was assessed by testing several commercial samples. Alternaria DNA was detected in 100% of spoiled tomato samples, 8% of tomato products, and 36.4% of cereal-based infant food samples analyzed.

Effect Of Cluster Intervention Strategy Combined with Targeted Nursing on Prevention of Ventilator-Associated Pneumonia in Icu Patients

2021 ◽  
Vol 7 (6) ◽  
pp. 6395-6401
Author(s):  
XueQin Li ◽  
XiuYing Chen
Keyword(s):  
Mechanical Ventilation ◽  
Detection Rate ◽  
Pathogenic Bacteria ◽  
Nursing Intervention ◽  
Apache Ii Score ◽  
Control Group ◽  
Study Group ◽  
Ventilator Associated Pneumonia ◽  
Icu Patients ◽  
Mechanical Ventilation Time

Background VAP is a common complication of ventilator maintenance therapy. The occurrence of VAP is related to many factors such as long duration of breathing, invasive operation, pollution of respiratory tubes and instruments, and low immunity of patients. The prevention of VAP in critically ill patients I the primary problem for clinical medical staff. Avoiding exogenous bacteria invading the respiratory tract and endogenous bacterial infection is the main method. Objective To investigate the value of optimized cluster nursing intervention combined with targeted nursing measures in reducing the incidence of ventilator-associated pneumonia (VAP) in patients with mechanical ventilation in intensive care unit (ICU). Methods 200 patients with mechanical ventilation in ICU of our institute from January 2017 to June 2020 were selected and randomly divided into study group and control group, with 100 cases in each group. The study group was treated with cluster nursing intervention combined with targeted nursing measures optimized by muItL criteria decision analysis method, and the control group was treated with targeted nursing measures. The incidence of VAP, the detection rate of pathogenic bacteria in sputum specimens and the effect of nursing execution were compared between the two groups. 200 patients were divided into VAP group and non-VAP group according to whether VAP occurred. Multivariate Logistic regression model analysis was used to explore the risk factors of VAP in AECOPD patients. Results A total of 4 strains were detected in the study group and 18 strains were detected in the control group. The detection rate of pathogenic bacteria in the study group was higher than that in the control group (y2=10.010, P=0.002<0.05). The incidence of VAP in the study group was 4.00% lower than 17.00% in the control group, and the difference was statistically significant (P<0.05). Compared with VAP group and non-VAP group, the proportion of patients with serum albumin<30g/L, diabetes mellitus rate, APACHE II score>15 points, tracheotomy rate and mechanical ventilation time≥5 days in VAP group were significantly higher than those in non-VAP group, which had statistical significance (P<0.05). The results of logistic regression model snowed that serum albumin ≥30g/L and optimized cluster nursing could effectively reduce the risk of VAP in ICU patients with mechanical ventilation (P<0.05). The risk of VAP in ICU patients with mechanical ventilation was increased by the combination of diabetes rate. APACHE II score≥15 points, tracheotomy and mechanical ventilation time ≥ 5 days (P<0.05). Conclusion The risk of VAP in ICU patients with mechanical ventilation is high, and the optimized cluster nursing intervention combined with targeted nursing measures can effectively reduce the incidence of VAP.

scholarly journals Antimicrobial Resistance in Bacterial Pathogens and Detection of Carbapenemases in Klebsiella pneumoniae Isolates from Hospital Wastewater

Antibiotics ◽  
2019 ◽  
Vol 8 (3) ◽  
pp. 85 ◽  
Author(s):  
Hercules Sakkas ◽  
Petros Bozidis ◽  
Afrodite Ilia ◽  
George Mpekoulis ◽  
Chrissanthy Papadopoulou
Keyword(s):  
Staphylococcus Aureus ◽  
Klebsiella Pneumoniae ◽  
Pathogenic Bacteria ◽  
Multidrug Resistant ◽  
Rapid Identification ◽  
Diffusion Method ◽  
Resistant Bacteria ◽  
Screening Methods ◽  
Extended Spectrum Beta Lactamase ◽  
Vancomycin Resistant

During a six-month period (October 2017–March 2018), the prevalence and susceptibility of important pathogenic bacteria isolated from 12 hospital raw sewage samples in North Western Greece was investigated. The samples were analyzed for methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant enterococci (VRE), extended-spectrum beta-lactamase (ESBL) producing Escherichia coli, carbapenemase-producing Klebsiella pneumoniae (CKP), and multidrug-resistant Pseudomonas aeruginosa. Antimicrobial susceptibility testing was performed using the agar diffusion method according to the recommendations of the Clinical and Laboratory Standards Institute. The diversity of carbapenemases harboring K. pneumoniae was examined by two phenotyping screening methods (modified Hodge test and combined disk test), a new immunochromatographic rapid assay (RESIST-4 O.K.N.V.) and a polymerase chain reaction (PCR). The results demonstrated the prevalence of MRSA, vancomycin-resistant Staphylococcus aureus (VRSA), VRE, and CKP in the examined hospital raw sewage samples. In addition, the aforementioned methods which are currently used in clinical laboratories for the rapid identification and detection of resistant bacteria and genes, performed sufficiently to provide reliable results in terms of accuracy and efficiency.

scholarly journals Compositional and Functional Characteristics of Swine Slurry Microbes through 16S rRNA Metagenomic Sequencing Approach

Animals ◽  
2020 ◽  
Vol 10 (8) ◽  
pp. 1372
Author(s):  
Himansu Kumar ◽  
Yu Jang ◽  
Kwangmin Kim ◽  
Junhyung Park ◽  
Min Jung ◽  
...  
Keyword(s):  
16S Rrna ◽  
Crop Production ◽  
Pathogenic Bacteria ◽  
Storage Time ◽  
Pig Slurry ◽  
Metagenomic Sequencing ◽  
Bacterial Populations ◽  
Linear Discriminant ◽  
Kegg Pathways ◽  
Microbial Biomarkers

Traditionally slurry is used as source of nitrogen, phosphorous, and potassium in bio fertilizers to improve crop production. However, poorly managed slurry causes a hazardous effect to the environment by producing greenhouse gases, causing the eutrophication of water bodies, and polluting the groundwater. It has been largely reported that the microbial presence in slurry causing a diverse effect on its storage and disposal system. However, the diversity of bacterial populations in pig slurries remains largely unexplored. Here we report the bacterial diversity present in the slurry from slurry pits, and the effect of storage time on bacterial population. We collected 42 samples from three different pig slurry pits, as three replicates from each one until the 14th week. We used the 16S rRNA, Quantitative Insights Into Microbial Ecology (QIIME) and Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt) protocols for the metagenomic downstream analysis. Taxonomic annotation using the Greengenes metagenomic database indicated that on an average 76.2% Firmicutes, 14.4% Bacteroidetes, 4.9% Proteobacteria, etc. microbial populations were present. Comparative microbial analysis showed that the population of Firmicutes decreased from the first to the 14th week, whereas the population of Bacteroidetes increased from the first to the 14th week. Through principal coordinate analysis (PCoA), (linear discriminant analysis effect size (LEfSe), and Pearson’s correlation analysis, we found microbial biomarkers according to the storage time point. All bacterial populations were well clustered according to the early, middle, and last weeks of storage. LEfSe showed that Actinobacteria, Lachnospiraceae, Ruminococcaceae, and Bacteroidia are dominantly present in first, seventh, ninth, and 14th week, respectively. Lachnospiraceae and Ruminococcaceae are ubiquitous gastrointestinal non-pathogenic bacteria. KEGG pathways, such as membrane transport, carbohydrate and amino acid metabolism, genetic replication and repair, were significant among all samples. Such a KEGG pathway may indicate the association between the host organism’s metabolic activity and the microbes present in the gastro intestinal tract (GIT).

Rapid identification and differentiation of fungal DNA in dermatological specimens by LightCycler PCR

2004 ◽  
Vol 53 (12) ◽  
pp. 1207-1214 ◽  
Author(s):  
Ralf Gutzmer ◽  
Susanne Mommert ◽  
Uta Küttler ◽  
Thomas Werfel ◽  
Alexander Kapp
Keyword(s):  
Skin Diseases ◽  
Rapid Identification ◽  
Diagnostic Information ◽  
Clinical Samples ◽  
Melting Points ◽  
Number Of Patients ◽  
Highly Sensitive ◽  
Fungal Dna ◽  
Pcr Method ◽  
Primer Sets

The aim was to develop a LightCycler PCR method for the rapid detection and differentiation of fungal DNA in dermatological specimens such as skin scales and skin swabs. LightCycler PCR assays were established for seven primer sets specific for fungal DNA. For each primer set LightCycler melting points were defined by amplification of DNA from 21 fungi and sensitivity was determined by amplification of serial dilutions of fungal DNA. A protocol was established that allows detection and differentiation of mould and yeast DNA with one highly sensitive PCR reaction by assessment of LightCycler melting points. Two subsequent LightCycler PCR reactions and one RFLP reaction allowed the differentiation of dermatophytes and non-dermatophyte moulds and the subclassification of yeasts. Analysis of clinical samples from 38 patients with fungal skin diseases provided conclusive new diagnostic information in 9/38 cases (23.7 %) by this PCR protocol that was not equally provided by direct microscopy and mycological culture. Thus the LightCycler PCR protocol established here represents a rapid diagnostic tool that aids in the diagnosis of fungal skin disease in a substantial number of patients.

scholarly journals Biodiversity and Classification of Lactococcal Phages

2006 ◽  
Vol 72 (6) ◽  
pp. 4338-4346 ◽  
Author(s):  
H�l�ne Deveau ◽  
Simon J. Labrie ◽  
Marie-Christine Chopin ◽  
Sylvain Moineau
Keyword(s):  
Dna Hybridization ◽  
Classification Scheme ◽  
Rapid Identification ◽  
New Classification ◽  
Sequence Analyses ◽  
Reference Center ◽  
Detection Tool ◽  
Pcr Product ◽  
Pcr Method

ABSTRACT For this study, an in-depth review of the classification of Lactococcus lactis phages was performed. Reference phages as well as unclassified phages from international collections were analyzed by stringent DNA-DNA hybridization studies, electron microscopy observations, and sequence analyses. A new classification scheme for lactococcal phages is proposed that reduces the current 12 groups to 8. However, two new phages (Q54 and 1706), which are unrelated to known lactococcal phages, may belong to new emerging groups. The multiplex PCR method currently used for the rapid identification of phages from the three main lactococcal groups (936, c2, and P335) was improved and tested against the other groups, none of which gave a PCR product, confirming the specificity of this detection tool. However, this method does not detect all members of the highly diverse P335 group. The lactococcal phages characterized here were deposited in the F�lix d'H�relle Reference Center for Bacterial Viruses and represent a highly diverse viral community from the dairy environment.

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