scholarly journals Rapid sequencing of MRSA direct from clinical plates in a routine microbiology laboratory

2019 ◽  
Vol 74 (8) ◽  
pp. 2153-2156 ◽  
Author(s):  
Beth Blane ◽  
Kathy E Raven ◽  
Danielle Leek ◽  
Nicholas Brown ◽  
Julian Parkhill ◽  
...  
Keyword(s):  
Dna Extraction ◽  
Clinical Laboratory ◽  
Culture Media ◽  
Direct Sequencing ◽  
Laboratory Culture ◽  
Outbreak Detection ◽  
Snp Analysis ◽  
University Hospitals ◽  
Quality Control Metrics ◽  
Clinical Culture

Abstract Background Routine sequencing of MRSA could bring about significant improvements to outbreak detection and investigation. Sequencing is commonly performed using DNA extracted from a pure culture, but overcoming the delay associated with this step could reduce the time to infection control interventions. Objectives To develop and evaluate rapid sequencing of MRSA using primary clinical cultures. Methods Patients with samples submitted to the clinical laboratory at the Cambridge University Hospitals NHS Foundation Trust from which MRSA was isolated were identified, the routine laboratory culture plates obtained and DNA extraction and sequencing performed. Results An evaluation of routine MRSA cultures from 30 patients demonstrated that direct sequencing from bacterial colonies picked from four different culture media was feasible. The 30 clinical MRSA isolates were sequenced on the day of plate retrieval over five runs and passed quality control metrics for sequencing depth and coverage. The maximum contamination detected using Kraken was 1.09% fragments, which were identified as Prevotella dentalis. The most common contaminants were other staphylococcal species (25 isolate sequences) and Burkholderia dolosa (11 isolate sequences). Core genome pairwise SNP analysis to identify clusters based on isolates that were ≤50 SNPs different was used to triage cases for further investigation. This identified three clusters, but more detailed genomic and epidemiological evaluation excluded an acute outbreak. Conclusions Rapid sequencing of MRSA from clinical culture plates is feasible and reduces the delay associated with purity culture prior to DNA extraction.

scholarly journals A common protocol for the simultaneous processing of multiple clinically relevant bacterial species for whole genome sequencing

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Kathy E. Raven ◽  
Sophia T. Girgis ◽  
Asha Akram ◽  
Beth Blane ◽  
Danielle Leek ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Dna Extraction ◽  
Genome Sequencing ◽  
Clinical Microbiology ◽  
Bacterial Species ◽  
Outbreak Detection ◽  
Whole Genome ◽  
Library Preparation ◽  
Simultaneous Processing ◽  
Antimicrobial Resistance Gene

AbstractWhole-genome sequencing is likely to become increasingly used by local clinical microbiology laboratories, where sequencing volume is low compared with national reference laboratories. Here, we describe a universal protocol for simultaneous DNA extraction and sequencing of numerous different bacterial species, allowing mixed species sequence runs to meet variable laboratory demand. We assembled test panels representing 20 clinically relevant bacterial species. The DNA extraction process used the QIAamp mini DNA kit, to which different combinations of reagents were added. Thereafter, a common protocol was used for library preparation and sequencing. The addition of lysostaphin, lysozyme or buffer ATL (a tissue lysis buffer) alone did not produce sufficient DNA for library preparation across the species tested. By contrast, lysozyme plus lysostaphin produced sufficient DNA across all 20 species. DNA from 15 of 20 species could be extracted from a 24-h culture plate, while the remainder required 48–72 h. The process demonstrated 100% reproducibility. Sequencing of the resulting DNA was used to recapitulate previous findings for species, outbreak detection, antimicrobial resistance gene detection and capsular type. This single protocol for simultaneous processing and sequencing of multiple bacterial species supports low volume and rapid turnaround time by local clinical microbiology laboratories.

scholarly journals A Pandemic Early Warning System Decision Analysis Concept Utilizing a Distributed Network of Air Samplers via Electrostatic Air Precipitation

2021 ◽  
Vol 11 (11) ◽  
pp. 5308
Author(s):  
Joseph J. Bango ◽  
Sophia A. Agostinelli ◽  
Makayla Maroney ◽  
Michael Dziekan ◽  
Ruba Deeb ◽  
...  
Keyword(s):  
Early Warning ◽  
Early Warning System ◽  
Pathogen Detection ◽  
Clinical Laboratory ◽  
Culture Media ◽  
Morphological Characteristics ◽  
Warning System ◽  
Ambient Air ◽  
Aerosol Sampling ◽  
Distributed Network

The COVID-19 pandemic has highlighted the need for improved airborne infectious disease monitoring capability. A key challenge is to develop a technology that captures pathogens for identification from ambient air. While pathogenic species vary significantly in size and shape, for effective airborne pathogen detection the target species must be selectively captured from aerosolized droplets. Captured pathogens must then be separated from the remaining aerosolized droplet content and characterized in real-time. While improvements have been made with clinical laboratory automated sorting in culture media based on morphological characteristics of cells, this application has not extended to aerosol samples containing bacteria, viruses, spores, or prions. This manuscript presents a strategy and a model for the development of an airborne pandemic early warning system using aerosol sampling. 

scholarly journals Methodological Approaches to DNA Authentication of Foods, Wines and Raw Materials for Their Production

Foods ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 595
Author(s):  
Aram G. Galstyan ◽  
Vladislav K. Semipyatniy ◽  
Irina Yu. Mikhailova ◽  
Khamid Kh. Gilmanov ◽  
Alana V. Bigaeva ◽  
...  
Keyword(s):  
Sample Preparation ◽  
Dna Extraction ◽  
Raw Materials ◽  
Direct Sequencing ◽  
Genetic Identification ◽  
Proteinase K ◽  
Vitis Vinifera L ◽  
Acid Extraction ◽  
Plant Component ◽  
Sensitivity Specificity

DNA authentication of wines is a process of verifying their authenticity by genetic identification of the main plant component. The sample preparation of experimental and commercial wines was carried out by precipitation of wine debris by centrifugation with preliminary exposure with precipitators and co-precipitators, including developed macro- and micro- volume methods applicable to white or red wines, using polyvinylpyrrolidone as a co-precipitator. Addition of 2-mercaptoethanol and proteinase K to the lysing solution made it possible to adapt the technology for DNA extraction from the precipitated wine debris. The additionally tested technique of DNA extraction from wine debris by dimethyl sulfoxide (DMSO) lysis had fewer stages and, consequently, a lower risk of contamination. The results of further testing of one of the designed primer pairs (UFGT-F1 and UFGT-R1) in conjunction with the tested methods of wine material sample preparation and nucleic acid extraction, showed the advantage in the given set of oligonucleotides over previously used ones in terms of sensitivity, specificity and reproducibility. The developing strategy for genetic identification of grape varieties and DNA authentication of wines produced from them based on direct sequencing of polymerase chain reaction (PCR) products is implemented by interpreting the detected polymorphic positions of variable Vitis vinifera L. UFGT gene locus with distribution and split into 13 UFGT gene-associated groups.

scholarly journals Leptin gene polymorphism of Ongole Grade cattle based on single nucleotide polymorphism

2018 ◽  
Vol 43 (4) ◽  
pp. 309
Author(s):  
N. Hilmia ◽  
D. Rahmat ◽  
D. Dudi
Keyword(s):  
Amino Acids ◽  
Single Nucleotide Polymorphism ◽  
Gene Polymorphism ◽  
Direct Sequencing ◽  
Snp Analysis ◽  
Nucleotide Polymorphism ◽  
Single Nucleotide ◽  
Exon 2 ◽  
Specific Allele ◽  
Polymerase Chain

Point mutation on exon 2 of leptin gene, which changes amino acid encoding from Arginine to Cysteine, may alters the physiological function of the leptin hormone. This study aimed to identify leptin gene polymorphism of Ongole Grade (OG) cattle based on Single Nucleotide Polymorphism (SNP). The DNA sample was taken from 48 head of OG cattle at Balai Pengembangan Perbibitan Ternak Sapi Potong(BPPT SP) Cijeungjing West Java, which was isolated from white blood cell using the high salt method. Amplification of DNA was done by Polymerase Chain Reaction (PCR), followed by direct sequencing to obtain nucleotide sequence. The SNP analysis was carried out from alignment of sequencing result using Bioedit and MEGA 5.2 program. The results indicated in exon 2 leptin gene of OG cattle there was one synonymous SNPs that did not changeamino acids Serine encoding on g.1025T >C/S17S, while two non synonymous SNPaltered amino acids encoding, those were g.1047C> T /R25C and g.1048G>A/R25H. Those mutations changed amino acids encoding from Arginine to Cysteine and Arginine to Histidine respectively.In OG cattle, the frequency of A allele (44.8%) was higher than C allele (33.3%) and T allele (21.9%). Six genotypes were also identified, i.e. AA (41.7%), CC (20.8%), CT (20.8%), CA(4.2%), TT (10.4%) and TA (2.1 %). Heterozigosity of OG cattle based on leptin gene was 0.65 that was a high category. The A allele was a specific allele on Indonesian local cattle.

scholarly journals Comparison of Three Skin Sampling Methods and Two Media for Culturing Malassezia Yeast

2020 ◽  
Vol 6 (4) ◽  
pp. 350
Author(s):  
Abdourahim Abdillah ◽  
Saber Khelaifia ◽  
Didier Raoult ◽  
Fadi Bittar ◽  
Stéphane Ranque
Keyword(s):  
Human Skin ◽  
Sampling Methods ◽  
Clinical Laboratory ◽  
Culture Media ◽  
Positive Culture ◽  
Laboratory Procedure ◽  
Study Results ◽  
Malassezia Spp ◽  
Routine Clinical Laboratory ◽  
Sterile Gauze

Malassezia is a lipid-dependent commensal yeast of the human skin. The different culture media and skin sampling methods used to grow these fastidious yeasts are a source of heterogeneity in culture-based epidemiological study results. This study aimed to compare the performances of three methods of skin sampling, and two culture media for the detection of Malassezia yeasts by culture from the human skin. Three skin sampling methods, namely sterile gauze, dry swab, and TranswabTM with transport medium, were applied on 10 healthy volunteers at 5 distinct body sites. Each sample was further inoculated onto either the novel FastFung medium or the reference Dixon agar for the detection of Malassezia spp. by culture. At least one colony of Malassezia spp. grew on 93/300 (31%) of the cultures, corresponding to 150 samplings. The positive culture rate was 67%, 18%, and 15% (P < 10−3), for samples collected with sterile gauze, TranswabTM, and dry swab, respectively. The positive culture rate was 62% and 38% (P < 0.003) by using the FastFung and the Dixon media, respectively. Our results showed that sterile gauze rubbing skin sampling followed by inoculation on FastFung medium should be implemented in the routine clinical laboratory procedure for Malassezia spp. cultivation.

Development and evaluation of a loop-mediated isothermal amplification assay for rapid detection of Mycoplasma pneumoniae

2005 ◽  
Vol 54 (11) ◽  
pp. 1037-1041 ◽  
Author(s):  
Ryoichi Saito ◽  
Yoshiki Misawa ◽  
Kyoji Moriya ◽  
Kazuhiko Koike ◽  
Kimiko Ubukata ◽  
...  
Keyword(s):  
Real Time ◽  
Mycoplasma Pneumoniae ◽  
Rapid Detection ◽  
Clinical Laboratory ◽  
Direct Sequencing ◽  
Bacterial Species ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Nasopharyngeal Swab ◽  
Loop Mediated Isothermal Amplification

A loop-mediated isothermal amplification (LAMP) assay for the rapid detection of Mycoplasma pneumoniae was developed and evaluated. The assay specifically amplified only M. pneumoniae sequences, and no cross-reactivity was observed for other Mycoplasma species or respiratory bacterial species. The detection limit for this assay was found to be 2 × 102 copies, corresponding to 2–20 colour changing units of M. pneumoniae in 1 h, as observed in a real-time turbidimeter and electrophoretic analysis. The accuracy of the LAMP reaction was confirmed by restriction endonuclease analysis as well as direct sequencing of the amplified product. The assay was applied to 95 nasopharyngeal swab samples collected from patients or from healthy individuals, and compared to a real-time PCR assay in-house. A concordance of 100 % was observed between the two assays. The LAMP assay is easy to perform, shows a rapid reaction and is inexpensive. It may therefore be applied in the routine diagnosis of M. pneumoniae infection in the clinical laboratory.

scholarly journals A Narrative Account of Nottingham University Hospitals NHS Trust Critical Care Response to the COVID-19 Pandemic, and Analysis of Outcomes

2020 ◽  
Author(s):  
Amar Javaid ◽  
Joel Swindin ◽  
Richard Greenhow ◽  
Marc Chikhani
Keyword(s):  
Critical Care ◽  
Mortality Rate ◽  
Clinical Laboratory ◽  
Management Strategies ◽  
Healthcare Services ◽  
Unexpected Finding ◽  
Case Review ◽  
University Hospitals ◽  
Retrospective Case ◽  
Multisystem Disease

Abstract BackgroundThe novel coronavirus SARS-CoV-2 and associated multisystem disease COVID-19 originated in Wuhan, China in December 2019. The resultant pandemic has resulted in an unprecedented strain on healthcare services around the world, including the greatest challenge seen by critical care medicine in modern times. This narrative illustrates the response of Nottingham University Hospitals NHS Trust critical care department to the crisis, including resource allocation and clinical management strategies and illustrates patient outcome for those admitted to critical care during the first pandemic wave.MethodsPatients admitted to critical care whom tested positive for SARS-CoV-2 between March-May 2020 were identified for retrospective case review as part of service evaluation. Clinical, laboratory and radiological data was collected. This included patient level data on ventilation and fluid balance.ResultsDuring the three period March-May 2020, retrospective case review showed 109 patients admitted to critical care with COVID-19. 88 (80.7%) of the 109 patients received mechanical ventilation, 21 (19.3%) patients received renal replacement therapy. Critical care mortality was 26 (23.9%) patients and hospital mortality was 27 (24.8%) of the 109 patients.ConclusionThis mortality rate was an unexpected finding based on early literature and help to provide a baseline for comparison hereafter. Commentary is provided for several aspects of care that may account for the low mortality rate and warrant further investigation in the future.

scholarly journals Laboratory Costs in the Context of Disease

2000 ◽  
Vol 46 (7) ◽  
pp. 967-975 ◽  
Author(s):  
Donald S Young ◽  
Bruce S Sachais ◽  
Leigh C Jefferies
Keyword(s):  
Clinical Laboratory ◽  
Diagnosis Related Groups ◽  
Point Of Care ◽  
Signs And Symptoms ◽  
Medical Patients ◽  
University Hospitals ◽  
Laboratory Cost ◽  
Transplant Patients ◽  
Lengths Of Stay ◽  
Hospital Patients

Abstract Background: To determine the contribution of laboratory costs to the overall costs of managing hospital patients with different diseases, we studied the costs of laboratory testing overall and in relation to the other costs incurred during hospitalization. Methods: We used a database developed by the University HealthSystems Consortium containing &gt;1 million patients in 60 University Hospitals with diseases included in 486 diagnosis-related groups (DRGs). Laboratory costs included in the database comprised those associated with testing in the clinical laboratory together with those incurred in point-of-care testing and anatomic pathology but not those involving blood products and their transfusion. Results: The mean laboratory costs to manage surgical patients were greater than those to manage medical patients in 19 of the 25 major diagnostic categories. The median laboratory costs for patients with liver transplants exceeded $8000, and the laboratory costs to support other organ transplants were among the highest. The highest proportion of total costs attributable to the laboratory was 18.3% for acute leukemia and kidney and urinary tract signs and symptoms, both in children. Laboratory costs were &lt;1.0% of the total costs for only 15 DRGs. The highest median daily laboratory cost, $416, was attributable to liver transplant patients. Several conditions had median laboratory costs less than $30 per day, in spite of lengths of stay that exceeded 10 days in some cases. Conclusions: Although laboratory costs generally average 6% of the total costs for surgical conditions and 9% of the total costs for medical conditions, there is considerable variability. In general, laboratory costs were relatively poorly correlated with total costs. However, observation of high daily laboratory costs for many DRGs suggests that reducing length of stay would reduce both laboratory and total costs.

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