A rapid, highly sensitive and culture-free detection of pathogens from blood by positive enrichment
Molecular diagnostics is a promising alternative to culture based methods for the detection of bloodstream infections, notably due to its overall lower turnaround time when starting directly from patient samples. Whole blood is usually the starting diagnostic sample in suspected bloodstream infectio...
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| Veröffentlicht in: | Journal of microbiological methods 2016-12, Vol.131, p.105-109 |
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| creator | Vutukuru, Manjula Ramya Sharma, Divya Khandige Ragavendar, MS Schmolke, Susanne Huang, Yiwei Gumbrecht, Walter Mitra, Nivedita |
| description | Molecular diagnostics is a promising alternative to culture based methods for the detection of bloodstream infections, notably due to its overall lower turnaround time when starting directly from patient samples. Whole blood is usually the starting diagnostic sample in suspected bloodstream infections. The detection of low concentrations of pathogens in blood using a molecular assay necessitates a fairly high starting volume of blood sample in the range of 5–10mL. This large volume of blood sample has a substantial accompanying human genomic content that interferes with pathogen detection. In this study, we have established a workflow using magnetic beads coated with Apolipoprotein H that makes it possible to concentrate pathogens from a 5.0mL whole blood sample, thereby enriching pathogens from whole blood background and also reducing the sample volume to ~200μL or less. We have also demonstrated that this method of enrichment allows detection of 1CFU/mL of Escherichia coli, Enterococcus gallinarum and Candida tropicalis from 5mL blood using quantitative PCR; a detection limit that is not possible in unenriched samples. The enrichment method demonstrated here took 30min to complete and can be easily integrated with various downstream molecular and microbiological techniques.
•Human genomic DNA background affects detection of microbes at |
| doi_str_mv | 10.1016/j.mimet.2016.10.008 |
| format | Article |
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•Human genomic DNA background affects detection of microbes at <10CFU/mL when present in blood•We demonstrate that positive enrichment using ApoH beads allows the detection of these microbes at 1CFU/mL in blood•An analytical sensitivity of 1CFU/mL was demonstrated with E. coli, Enterococcus gallinarum and Candida albicans•This method also decreases the sample volume significantly thus simplifying the subsequent nucleic acid extraction process</description><identifier>ISSN: 0167-7012</identifier><identifier>EISSN: 1872-8359</identifier><identifier>DOI: 10.1016/j.mimet.2016.10.008</identifier><identifier>PMID: 27765617</identifier><language>eng</language><publisher>Netherlands: Elsevier B.V</publisher><subject><![CDATA[Apolipoprotein H ; Bacteria - classification ; Bacteria - genetics ; Bacteria - isolation & purification ; Bacteria - pathogenicity ; beta 2-Glycoprotein I - administration & dosage ; Blood - microbiology ; Blood stream infections ; Blood-Borne Pathogens - isolation & purification ; Candida tropicalis ; Candida tropicalis - genetics ; Candida tropicalis - isolation & purification ; Cells, Cultured ; Colony Count, Microbial - methods ; Culture-free detection ; DNA, Bacterial - blood ; DNA, Fungal - blood ; Enterococcus - genetics ; Enterococcus - isolation & purification ; Enterococcus gallinarum ; Escherichia coli ; Escherichia coli - genetics ; Escherichia coli - isolation & purification ; Fungi - classification ; Fungi - genetics ; Fungi - isolation & purification ; Fungi - pathogenicity ; Genome, Human ; Humans ; Limit of Detection ; Microbiological Techniques - methods ; Pathogen enrichment ; Pathology, Molecular - methods ; Quantitative PCR ; Real-Time Polymerase Chain Reaction - methods ; Sensitivity and Specificity ; Sepsis ; Sepsis - blood ; Sepsis - diagnosis ; Sepsis - microbiology ; Time Factors]]></subject><ispartof>Journal of microbiological methods, 2016-12, Vol.131, p.105-109</ispartof><rights>2016 Elsevier B.V.</rights><rights>Copyright © 2016 Elsevier B.V. All rights reserved.</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c392t-60174a59b7bb72d89e909d1a16beb56892d3b44cf29461da2bdae0a01326ecdc3</citedby><cites>FETCH-LOGICAL-c392t-60174a59b7bb72d89e909d1a16beb56892d3b44cf29461da2bdae0a01326ecdc3</cites><orcidid>0000-0003-3799-5681</orcidid></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktohtml>$$Uhttps://dx.doi.org/10.1016/j.mimet.2016.10.008$$EHTML$$P50$$Gelsevier$$H</linktohtml><link.rule.ids>314,780,784,3550,27924,27925,45995</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/27765617$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Vutukuru, Manjula Ramya</creatorcontrib><creatorcontrib>Sharma, Divya Khandige</creatorcontrib><creatorcontrib>Ragavendar, MS</creatorcontrib><creatorcontrib>Schmolke, Susanne</creatorcontrib><creatorcontrib>Huang, Yiwei</creatorcontrib><creatorcontrib>Gumbrecht, Walter</creatorcontrib><creatorcontrib>Mitra, Nivedita</creatorcontrib><title>A rapid, highly sensitive and culture-free detection of pathogens from blood by positive enrichment</title><title>Journal of microbiological methods</title><addtitle>J Microbiol Methods</addtitle><description>Molecular diagnostics is a promising alternative to culture based methods for the detection of bloodstream infections, notably due to its overall lower turnaround time when starting directly from patient samples. Whole blood is usually the starting diagnostic sample in suspected bloodstream infections. The detection of low concentrations of pathogens in blood using a molecular assay necessitates a fairly high starting volume of blood sample in the range of 5–10mL. This large volume of blood sample has a substantial accompanying human genomic content that interferes with pathogen detection. In this study, we have established a workflow using magnetic beads coated with Apolipoprotein H that makes it possible to concentrate pathogens from a 5.0mL whole blood sample, thereby enriching pathogens from whole blood background and also reducing the sample volume to ~200μL or less. We have also demonstrated that this method of enrichment allows detection of 1CFU/mL of Escherichia coli, Enterococcus gallinarum and Candida tropicalis from 5mL blood using quantitative PCR; a detection limit that is not possible in unenriched samples. The enrichment method demonstrated here took 30min to complete and can be easily integrated with various downstream molecular and microbiological techniques.
•Human genomic DNA background affects detection of microbes at <10CFU/mL when present in blood•We demonstrate that positive enrichment using ApoH beads allows the detection of these microbes at 1CFU/mL in blood•An analytical sensitivity of 1CFU/mL was demonstrated with E. coli, Enterococcus gallinarum and Candida albicans•This method also decreases the sample volume significantly thus simplifying the subsequent nucleic acid extraction process</description><subject>Apolipoprotein H</subject><subject>Bacteria - classification</subject><subject>Bacteria - genetics</subject><subject>Bacteria - isolation & purification</subject><subject>Bacteria - pathogenicity</subject><subject>beta 2-Glycoprotein I - administration & dosage</subject><subject>Blood - microbiology</subject><subject>Blood stream infections</subject><subject>Blood-Borne Pathogens - isolation & purification</subject><subject>Candida tropicalis</subject><subject>Candida tropicalis - genetics</subject><subject>Candida tropicalis - isolation & purification</subject><subject>Cells, Cultured</subject><subject>Colony Count, Microbial - methods</subject><subject>Culture-free detection</subject><subject>DNA, Bacterial - blood</subject><subject>DNA, Fungal - blood</subject><subject>Enterococcus - genetics</subject><subject>Enterococcus - isolation & purification</subject><subject>Enterococcus gallinarum</subject><subject>Escherichia coli</subject><subject>Escherichia coli - genetics</subject><subject>Escherichia coli - isolation & purification</subject><subject>Fungi - classification</subject><subject>Fungi - genetics</subject><subject>Fungi - isolation & purification</subject><subject>Fungi - pathogenicity</subject><subject>Genome, Human</subject><subject>Humans</subject><subject>Limit of Detection</subject><subject>Microbiological Techniques - methods</subject><subject>Pathogen enrichment</subject><subject>Pathology, Molecular - methods</subject><subject>Quantitative PCR</subject><subject>Real-Time Polymerase Chain Reaction - methods</subject><subject>Sensitivity and Specificity</subject><subject>Sepsis</subject><subject>Sepsis - blood</subject><subject>Sepsis - diagnosis</subject><subject>Sepsis - microbiology</subject><subject>Time Factors</subject><issn>0167-7012</issn><issn>1872-8359</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2016</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqNkU1v1DAQhi0EotvCL0BCPnJoFn8kdnzgUFWUIlXiQs-WPyZdr5I42E6l_fd4uwtHxGk0M887I70vQh8o2VJCxef9dgoTlC2rTZ1sCelfoQ3tJWt63qnXaFMXspGEsgt0mfOeENrxtn-LLpiUohNUbpC7wckswV_jXXjajQecYc6hhGfAZvbYrWNZEzRDAsAeCrgS4ozjgBdTdvGpwnhIccJ2jNFje8BLPMthTsHtJpjLO_RmMGOG9-d6hR7vvv68vW8efnz7fnvz0DiuWGkEobI1nbLSWsl8r0AR5amhwoLtRK-Y57Zt3cBUK6g3zHoDxBDKmQDnHb9Cn053lxR_rZCLnkJ2MI5mhrhmTXvRcy6pJP-B8q6jquOiovyEuhRzTjDoJYXJpIOmRB-D0Hv9EoQ-BnEc1iCq6uP5wWon8H81f5yvwJcTANWR5wBJZxdgduBDqi5rH8M_H_wGyi6bYg</recordid><startdate>201612</startdate><enddate>201612</enddate><creator>Vutukuru, Manjula Ramya</creator><creator>Sharma, Divya Khandige</creator><creator>Ragavendar, MS</creator><creator>Schmolke, Susanne</creator><creator>Huang, Yiwei</creator><creator>Gumbrecht, Walter</creator><creator>Mitra, Nivedita</creator><general>Elsevier B.V</general><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7X8</scope><scope>7T7</scope><scope>8FD</scope><scope>C1K</scope><scope>FR3</scope><scope>P64</scope><orcidid>https://orcid.org/0000-0003-3799-5681</orcidid></search><sort><creationdate>201612</creationdate><title>A rapid, highly sensitive and culture-free detection of pathogens from blood by positive enrichment</title><author>Vutukuru, Manjula Ramya ; Sharma, Divya Khandige ; Ragavendar, MS ; Schmolke, Susanne ; Huang, Yiwei ; Gumbrecht, Walter ; Mitra, Nivedita</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c392t-60174a59b7bb72d89e909d1a16beb56892d3b44cf29461da2bdae0a01326ecdc3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2016</creationdate><topic>Apolipoprotein H</topic><topic>Bacteria - classification</topic><topic>Bacteria - genetics</topic><topic>Bacteria - isolation & purification</topic><topic>Bacteria - pathogenicity</topic><topic>beta 2-Glycoprotein I - administration & dosage</topic><topic>Blood - microbiology</topic><topic>Blood stream infections</topic><topic>Blood-Borne Pathogens - isolation & purification</topic><topic>Candida tropicalis</topic><topic>Candida tropicalis - genetics</topic><topic>Candida tropicalis - isolation & purification</topic><topic>Cells, Cultured</topic><topic>Colony Count, Microbial - methods</topic><topic>Culture-free detection</topic><topic>DNA, Bacterial - blood</topic><topic>DNA, Fungal - blood</topic><topic>Enterococcus - genetics</topic><topic>Enterococcus - isolation & purification</topic><topic>Enterococcus gallinarum</topic><topic>Escherichia coli</topic><topic>Escherichia coli - genetics</topic><topic>Escherichia coli - isolation & purification</topic><topic>Fungi - classification</topic><topic>Fungi - genetics</topic><topic>Fungi - isolation & purification</topic><topic>Fungi - pathogenicity</topic><topic>Genome, Human</topic><topic>Humans</topic><topic>Limit of Detection</topic><topic>Microbiological Techniques - methods</topic><topic>Pathogen enrichment</topic><topic>Pathology, Molecular - methods</topic><topic>Quantitative PCR</topic><topic>Real-Time Polymerase Chain Reaction - methods</topic><topic>Sensitivity and Specificity</topic><topic>Sepsis</topic><topic>Sepsis - blood</topic><topic>Sepsis - diagnosis</topic><topic>Sepsis - microbiology</topic><topic>Time Factors</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Vutukuru, Manjula Ramya</creatorcontrib><creatorcontrib>Sharma, Divya Khandige</creatorcontrib><creatorcontrib>Ragavendar, MS</creatorcontrib><creatorcontrib>Schmolke, Susanne</creatorcontrib><creatorcontrib>Huang, Yiwei</creatorcontrib><creatorcontrib>Gumbrecht, Walter</creatorcontrib><creatorcontrib>Mitra, Nivedita</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>MEDLINE - Academic</collection><collection>Industrial and Applied Microbiology Abstracts (Microbiology A)</collection><collection>Technology Research Database</collection><collection>Environmental Sciences and Pollution Management</collection><collection>Engineering Research Database</collection><collection>Biotechnology and BioEngineering Abstracts</collection><jtitle>Journal of microbiological methods</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Vutukuru, Manjula Ramya</au><au>Sharma, Divya Khandige</au><au>Ragavendar, MS</au><au>Schmolke, Susanne</au><au>Huang, Yiwei</au><au>Gumbrecht, Walter</au><au>Mitra, Nivedita</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>A rapid, highly sensitive and culture-free detection of pathogens from blood by positive enrichment</atitle><jtitle>Journal of microbiological methods</jtitle><addtitle>J Microbiol Methods</addtitle><date>2016-12</date><risdate>2016</risdate><volume>131</volume><spage>105</spage><epage>109</epage><pages>105-109</pages><issn>0167-7012</issn><eissn>1872-8359</eissn><abstract>Molecular diagnostics is a promising alternative to culture based methods for the detection of bloodstream infections, notably due to its overall lower turnaround time when starting directly from patient samples. Whole blood is usually the starting diagnostic sample in suspected bloodstream infections. The detection of low concentrations of pathogens in blood using a molecular assay necessitates a fairly high starting volume of blood sample in the range of 5–10mL. This large volume of blood sample has a substantial accompanying human genomic content that interferes with pathogen detection. In this study, we have established a workflow using magnetic beads coated with Apolipoprotein H that makes it possible to concentrate pathogens from a 5.0mL whole blood sample, thereby enriching pathogens from whole blood background and also reducing the sample volume to ~200μL or less. We have also demonstrated that this method of enrichment allows detection of 1CFU/mL of Escherichia coli, Enterococcus gallinarum and Candida tropicalis from 5mL blood using quantitative PCR; a detection limit that is not possible in unenriched samples. The enrichment method demonstrated here took 30min to complete and can be easily integrated with various downstream molecular and microbiological techniques.
•Human genomic DNA background affects detection of microbes at <10CFU/mL when present in blood•We demonstrate that positive enrichment using ApoH beads allows the detection of these microbes at 1CFU/mL in blood•An analytical sensitivity of 1CFU/mL was demonstrated with E. coli, Enterococcus gallinarum and Candida albicans•This method also decreases the sample volume significantly thus simplifying the subsequent nucleic acid extraction process</abstract><cop>Netherlands</cop><pub>Elsevier B.V</pub><pmid>27765617</pmid><doi>10.1016/j.mimet.2016.10.008</doi><tpages>5</tpages><orcidid>https://orcid.org/0000-0003-3799-5681</orcidid></addata></record> |
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| ispartof | Journal of microbiological methods, 2016-12, Vol.131, p.105-109 |
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| language | eng |
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| subjects | Apolipoprotein H Bacteria - classification Bacteria - genetics Bacteria - isolation & purification Bacteria - pathogenicity beta 2-Glycoprotein I - administration & dosage Blood - microbiology Blood stream infections Blood-Borne Pathogens - isolation & purification Candida tropicalis Candida tropicalis - genetics Candida tropicalis - isolation & purification Cells, Cultured Colony Count, Microbial - methods Culture-free detection DNA, Bacterial - blood DNA, Fungal - blood Enterococcus - genetics Enterococcus - isolation & purification Enterococcus gallinarum Escherichia coli Escherichia coli - genetics Escherichia coli - isolation & purification Fungi - classification Fungi - genetics Fungi - isolation & purification Fungi - pathogenicity Genome, Human Humans Limit of Detection Microbiological Techniques - methods Pathogen enrichment Pathology, Molecular - methods Quantitative PCR Real-Time Polymerase Chain Reaction - methods Sensitivity and Specificity Sepsis Sepsis - blood Sepsis - diagnosis Sepsis - microbiology Time Factors |
| title | A rapid, highly sensitive and culture-free detection of pathogens from blood by positive enrichment |
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