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660. Extraction-Free 16S Ribosomal RNA (rRNA) Gene Amplification and Sequencing from Resected Cardiac Implantable Electronic Device (CIED) Sonicate Fluid
BACKGROUND: We recently demonstrated that 16S rRNA PCR/sequencing performed on biofilms dislodged from extracted CIEDs into a salt solution—referred to as “sonicate fluid’ (SF)—may be used to detect pathogens in culture-negative CIED infection (Clinical Infectious Diseases, ciz266, doi:10.1093/cid/c...
Autores principales: | , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Oxford University Press
2019
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6811131/ http://dx.doi.org/10.1093/ofid/ofz360.728 |
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author | Esquer Garrigos, Zerelda Sohail, Muhammad R Greenwood-Quaintance, Kerryl Cunningham, Scott A Vijayvargiya, Prakhar Patel, Robin Patel, Robin |
author_facet | Esquer Garrigos, Zerelda Sohail, Muhammad R Greenwood-Quaintance, Kerryl Cunningham, Scott A Vijayvargiya, Prakhar Patel, Robin Patel, Robin |
author_sort | Esquer Garrigos, Zerelda |
collection | PubMed |
description | BACKGROUND: We recently demonstrated that 16S rRNA PCR/sequencing performed on biofilms dislodged from extracted CIEDs into a salt solution—referred to as “sonicate fluid’ (SF)—may be used to detect pathogens in culture-negative CIED infection (Clinical Infectious Diseases, ciz266, doi:10.1093/cid/ciz266). The method we described included a DNA extraction/purification step, which can be time consuming and costly. Here, we evaluated an extraction-free approach to 16S rRNA gene PCR/sequencing. METHODS: 28 SF samples derived from explanted clinically-infected CIEDs were tested. Cases were categorized as “culture-positive” (C-P) if ≥20 cfu/10 mL were recovered, and as “culture-negative” (C-N), if <20 cfu/10 mL were detected in SF culture. The extraction-free method consisted of a single step of lysis at 1,000 rpm, 95°C for 5 minutes using an Eppendorf ThermoMixer®. DNA extraction (the comparator method) was performed using the ZymoBIOMICS(TM) Kit with modifications. Samples were processed using both methods, followed by amplification of the 16S rRNA gene and bidirectional Sanger sequencing. Crossing points (CPs) generated by the two approaches were compared. Organisms detected by the two PCR methods were compared with those detected with culture. RESULTS: Of the 28 samples tested, 13 were C-N and 15 C-P. The extraction-free method generated an amplicon in 13/15 C-P cases, with CPs ranging from 26 to 36 cycles vs. 100% (15/15) detected with the DNA extraction method and Cps of 19 to 32. Usable sequence length for the extraction-free method was of 359 (interquartile range, 307–390) vs. 390 (interquartile range, 308–396) base pairs with DNA extraction. Genus-level concordance between bacteria detected by culture in C-P samples and those found using the extraction-free and extraction methods was 92% (12/13) and 93% (14/15), respectively. Bacteria were detected by the extraction method in 2/13 C-N specimens, with none detected with an extraction-free method. CONCLUSION: The described extraction-free method may be suitable for testing SF derived from CIEDs using 16S rRNA gene PCR/sequencing, saving time and cost. More studies are needed to establish clear cutoffs for interpretation of results and to assess for PCR inhibitors in the studied specimen-type. DISCLOSURES: Robin Patel, MD, ASM and IDSA: Other Financial or Material Support, Travel reimbursement, editor’s stipends; CD Diagnostics, Merck, Hutchison Biofilm Medical Solutions, Accelerate Diagnostics, ContraFect, TenNor Therapeutics Limited, Shionogi: Grant/Research Support; Curetis, Specific Technologies, NextGen Diagnostics, PathoQuest, Qvella: Consultant; NBME, Up-to-Date, the Infectious Diseases Board Review Course: Honorarium recipient, Other Financial or Material Support; Patent on Bordetella pertussis/parapertussis PCR issued, a patent on a device/method for sonication with royalties paid by Samsung to Mayo Clinic, and a patent on an anti-biofilm substance issued: Other Financial or Material Support, Patents. |
format | Online Article Text |
id | pubmed-6811131 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2019 |
publisher | Oxford University Press |
record_format | MEDLINE/PubMed |
spelling | pubmed-68111312019-10-28 660. Extraction-Free 16S Ribosomal RNA (rRNA) Gene Amplification and Sequencing from Resected Cardiac Implantable Electronic Device (CIED) Sonicate Fluid Esquer Garrigos, Zerelda Sohail, Muhammad R Greenwood-Quaintance, Kerryl Cunningham, Scott A Vijayvargiya, Prakhar Patel, Robin Patel, Robin Open Forum Infect Dis Abstracts BACKGROUND: We recently demonstrated that 16S rRNA PCR/sequencing performed on biofilms dislodged from extracted CIEDs into a salt solution—referred to as “sonicate fluid’ (SF)—may be used to detect pathogens in culture-negative CIED infection (Clinical Infectious Diseases, ciz266, doi:10.1093/cid/ciz266). The method we described included a DNA extraction/purification step, which can be time consuming and costly. Here, we evaluated an extraction-free approach to 16S rRNA gene PCR/sequencing. METHODS: 28 SF samples derived from explanted clinically-infected CIEDs were tested. Cases were categorized as “culture-positive” (C-P) if ≥20 cfu/10 mL were recovered, and as “culture-negative” (C-N), if <20 cfu/10 mL were detected in SF culture. The extraction-free method consisted of a single step of lysis at 1,000 rpm, 95°C for 5 minutes using an Eppendorf ThermoMixer®. DNA extraction (the comparator method) was performed using the ZymoBIOMICS(TM) Kit with modifications. Samples were processed using both methods, followed by amplification of the 16S rRNA gene and bidirectional Sanger sequencing. Crossing points (CPs) generated by the two approaches were compared. Organisms detected by the two PCR methods were compared with those detected with culture. RESULTS: Of the 28 samples tested, 13 were C-N and 15 C-P. The extraction-free method generated an amplicon in 13/15 C-P cases, with CPs ranging from 26 to 36 cycles vs. 100% (15/15) detected with the DNA extraction method and Cps of 19 to 32. Usable sequence length for the extraction-free method was of 359 (interquartile range, 307–390) vs. 390 (interquartile range, 308–396) base pairs with DNA extraction. Genus-level concordance between bacteria detected by culture in C-P samples and those found using the extraction-free and extraction methods was 92% (12/13) and 93% (14/15), respectively. Bacteria were detected by the extraction method in 2/13 C-N specimens, with none detected with an extraction-free method. CONCLUSION: The described extraction-free method may be suitable for testing SF derived from CIEDs using 16S rRNA gene PCR/sequencing, saving time and cost. More studies are needed to establish clear cutoffs for interpretation of results and to assess for PCR inhibitors in the studied specimen-type. DISCLOSURES: Robin Patel, MD, ASM and IDSA: Other Financial or Material Support, Travel reimbursement, editor’s stipends; CD Diagnostics, Merck, Hutchison Biofilm Medical Solutions, Accelerate Diagnostics, ContraFect, TenNor Therapeutics Limited, Shionogi: Grant/Research Support; Curetis, Specific Technologies, NextGen Diagnostics, PathoQuest, Qvella: Consultant; NBME, Up-to-Date, the Infectious Diseases Board Review Course: Honorarium recipient, Other Financial or Material Support; Patent on Bordetella pertussis/parapertussis PCR issued, a patent on a device/method for sonication with royalties paid by Samsung to Mayo Clinic, and a patent on an anti-biofilm substance issued: Other Financial or Material Support, Patents. Oxford University Press 2019-10-23 /pmc/articles/PMC6811131/ http://dx.doi.org/10.1093/ofid/ofz360.728 Text en © The Author(s) 2019. Published by Oxford University Press on behalf of Infectious Diseases Society of America. http://creativecommons.org/licenses/by-nc-nd/4.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs licence (http://creativecommons.org/licenses/by-nc-nd/4.0/), which permits non-commercial reproduction and distribution of the work, in any medium, provided the original work is not altered or transformed in any way, and that the work is properly cited. For commercial re-use, please contact journals.permissions@oup.com |
spellingShingle | Abstracts Esquer Garrigos, Zerelda Sohail, Muhammad R Greenwood-Quaintance, Kerryl Cunningham, Scott A Vijayvargiya, Prakhar Patel, Robin Patel, Robin 660. Extraction-Free 16S Ribosomal RNA (rRNA) Gene Amplification and Sequencing from Resected Cardiac Implantable Electronic Device (CIED) Sonicate Fluid |
title | 660. Extraction-Free 16S Ribosomal RNA (rRNA) Gene Amplification and Sequencing from Resected Cardiac Implantable Electronic Device (CIED) Sonicate Fluid |
title_full | 660. Extraction-Free 16S Ribosomal RNA (rRNA) Gene Amplification and Sequencing from Resected Cardiac Implantable Electronic Device (CIED) Sonicate Fluid |
title_fullStr | 660. Extraction-Free 16S Ribosomal RNA (rRNA) Gene Amplification and Sequencing from Resected Cardiac Implantable Electronic Device (CIED) Sonicate Fluid |
title_full_unstemmed | 660. Extraction-Free 16S Ribosomal RNA (rRNA) Gene Amplification and Sequencing from Resected Cardiac Implantable Electronic Device (CIED) Sonicate Fluid |
title_short | 660. Extraction-Free 16S Ribosomal RNA (rRNA) Gene Amplification and Sequencing from Resected Cardiac Implantable Electronic Device (CIED) Sonicate Fluid |
title_sort | 660. extraction-free 16s ribosomal rna (rrna) gene amplification and sequencing from resected cardiac implantable electronic device (cied) sonicate fluid |
topic | Abstracts |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6811131/ http://dx.doi.org/10.1093/ofid/ofz360.728 |
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