Demonstration of a fast and easy sample-to-answer protocol for tuberculosis screening in point-of-care settings: A proof of concept study
We sought to develop a smooth and low cost sample preparation and DNA extraction protocol, streamlined with a ready-to-use qPCR in a portable instrument to overcome some of the existing hurdles. Several solutions were evaluated as to their ability to liquefy a mucin-based matrix. Each liquefied matr...
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| Veröffentlicht in: | PloS one 2020-12, Vol.15 (12), p.e0242408-e0242408 |
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| creator | Ali, Nasir Bello, Graziele Lima Rossetti, Maria Lúcia Rosa Krieger, Marco Aurelio Costa, Alexandre Dias Tavares |
| description | We sought to develop a smooth and low cost sample preparation and DNA extraction protocol, streamlined with a ready-to-use qPCR in a portable instrument to overcome some of the existing hurdles. Several solutions were evaluated as to their ability to liquefy a mucin-based matrix. Each liquefied matrix, supplemented with either Mycobacterium tuberculosis (MTB) H37Rv strain DNA or intact cells, was aliquoted onto a filter paper embedded with solubilizing agents, and was subsequently dried up. Most of the nucleic acids, including genomic DNA from the bacilli and the host, binds to the filter paper. Next, several protocols were evaluated to elute the DNA from the paper, using qPCR to detect the insertion sequence IS6110, a M. tuberculosis complex genomic marker. The limit of detection (LOD) of the best protocol was then evaluated using parallel seeding and colony counting. The protocol was also evaluated using seventeen sputum samples, previously characterized by the GeneXpert or culture. Two instruments (the ABI7500 Standard and the Q3-Plus system) and two reagents storage formats (frozen or ready-to-use) were evaluated. Solutions containing guanidine isothiocyanate exerted the best liquefying effect on the mucin-based matrix extracted from one 6-mm punches, followed by a brief incubation at 95°C. The resulting DNA contained impurities, but a simple 1:10 dilution elicited the detection of MTB and human genomic targets. The described protocol presented an apparent LOD of 02 CFU/mL of MTB. Challenging the protocol with previously characterized samples showed substantial agreement with GeneXpert MTB/RIF results (sensitivity of 90%, agreement of 88.9%, kappa coefficient of 0.77), and moderate agreement with culture results (sensitivity of 100%, agreement of 78.9%, kappa coefficient of 0.58). This work presents a sensitive proof-of-concept protocol for sputum liquefaction and decontamination followed by a simple DNA extraction procedure, in which the extraction steps are streamlined with a ready-to-use qPCR in a portable instrument that can be employed in low infrastructure settings. |
| doi_str_mv | 10.1371/journal.pone.0242408 |
| format | Article |
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Several solutions were evaluated as to their ability to liquefy a mucin-based matrix. Each liquefied matrix, supplemented with either Mycobacterium tuberculosis (MTB) H37Rv strain DNA or intact cells, was aliquoted onto a filter paper embedded with solubilizing agents, and was subsequently dried up. Most of the nucleic acids, including genomic DNA from the bacilli and the host, binds to the filter paper. Next, several protocols were evaluated to elute the DNA from the paper, using qPCR to detect the insertion sequence IS6110, a M. tuberculosis complex genomic marker. The limit of detection (LOD) of the best protocol was then evaluated using parallel seeding and colony counting. The protocol was also evaluated using seventeen sputum samples, previously characterized by the GeneXpert or culture. Two instruments (the ABI7500 Standard and the Q3-Plus system) and two reagents storage formats (frozen or ready-to-use) were evaluated. Solutions containing guanidine isothiocyanate exerted the best liquefying effect on the mucin-based matrix extracted from one 6-mm punches, followed by a brief incubation at 95°C. The resulting DNA contained impurities, but a simple 1:10 dilution elicited the detection of MTB and human genomic targets. The described protocol presented an apparent LOD of 02 CFU/mL of MTB. Challenging the protocol with previously characterized samples showed substantial agreement with GeneXpert MTB/RIF results (sensitivity of 90%, agreement of 88.9%, kappa coefficient of 0.77), and moderate agreement with culture results (sensitivity of 100%, agreement of 78.9%, kappa coefficient of 0.58). This work presents a sensitive proof-of-concept protocol for sputum liquefaction and decontamination followed by a simple DNA extraction procedure, in which the extraction steps are streamlined with a ready-to-use qPCR in a portable instrument that can be employed in low infrastructure settings.</description><identifier>ISSN: 1932-6203</identifier><identifier>EISSN: 1932-6203</identifier><identifier>DOI: 10.1371/journal.pone.0242408</identifier><identifier>PMID: 33315885</identifier><language>eng</language><publisher>United States: Public Library of Science</publisher><subject>Bacilli ; Biology and Life Sciences ; Brazil ; Cell culture ; Costs ; Culture ; Decontamination ; Deoxyribonucleic acid ; Diagnosis ; Dilution ; DNA ; DNA, Bacterial - isolation & purification ; Filter paper ; Genomics ; Guanidine ; Humans ; Impurities ; Isothiocyanate ; Leprosy ; Limit of Detection ; Liquefaction ; Mass Screening - economics ; Mass Screening - instrumentation ; Mass Screening - methods ; Medical diagnosis ; Medical screening ; Medical tests ; Medicine and Health Sciences ; Methods ; Microscopy ; Mucin ; Mycobacterium tuberculosis - genetics ; Mycobacterium tuberculosis - isolation & purification ; Nucleic acids ; Nucleotide sequence ; Physical Sciences ; Point-of-Care Systems ; Proof of Concept Study ; Punches ; Reagents ; Real-Time Polymerase Chain Reaction - economics ; Real-Time Polymerase Chain Reaction - instrumentation ; Real-Time Polymerase Chain Reaction - methods ; Research and analysis methods ; Sample preparation ; Sensitivity ; Specimen Handling - economics ; Specimen Handling - methods ; Sputum ; Sputum - microbiology ; Tuberculosis ; Tuberculosis, Pulmonary - diagnosis ; Tuberculosis, Pulmonary - microbiology ; Viscosity</subject><ispartof>PloS one, 2020-12, Vol.15 (12), p.e0242408-e0242408</ispartof><rights>COPYRIGHT 2020 Public Library of Science</rights><rights>2020 Ali et al. This is an open access article distributed under the terms of the Creative Commons Attribution License: http://creativecommons.org/licenses/by/4.0/ (the “License”), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License.</rights><rights>2020 Ali et al 2020 Ali et al</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c692t-e384ffc073a96021e568292cc6103811741d3393486c5362e79e76c134cecee63</citedby><cites>FETCH-LOGICAL-c692t-e384ffc073a96021e568292cc6103811741d3393486c5362e79e76c134cecee63</cites><orcidid>0000-0001-8434-2152</orcidid></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC7735633/pdf/$$EPDF$$P50$$Gpubmedcentral$$Hfree_for_read</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC7735633/$$EHTML$$P50$$Gpubmedcentral$$Hfree_for_read</linktohtml><link.rule.ids>230,314,724,777,781,861,882,2096,2915,23847,27905,27906,53772,53774,79349,79350</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/33315885$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><contributor>Hasnain, Seyed Ehtesham</contributor><creatorcontrib>Ali, Nasir</creatorcontrib><creatorcontrib>Bello, Graziele Lima</creatorcontrib><creatorcontrib>Rossetti, Maria Lúcia Rosa</creatorcontrib><creatorcontrib>Krieger, Marco Aurelio</creatorcontrib><creatorcontrib>Costa, Alexandre Dias Tavares</creatorcontrib><title>Demonstration of a fast and easy sample-to-answer protocol for tuberculosis screening in point-of-care settings: A proof of concept study</title><title>PloS one</title><addtitle>PLoS One</addtitle><description>We sought to develop a smooth and low cost sample preparation and DNA extraction protocol, streamlined with a ready-to-use qPCR in a portable instrument to overcome some of the existing hurdles. 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Solutions containing guanidine isothiocyanate exerted the best liquefying effect on the mucin-based matrix extracted from one 6-mm punches, followed by a brief incubation at 95°C. The resulting DNA contained impurities, but a simple 1:10 dilution elicited the detection of MTB and human genomic targets. The described protocol presented an apparent LOD of 02 CFU/mL of MTB. Challenging the protocol with previously characterized samples showed substantial agreement with GeneXpert MTB/RIF results (sensitivity of 90%, agreement of 88.9%, kappa coefficient of 0.77), and moderate agreement with culture results (sensitivity of 100%, agreement of 78.9%, kappa coefficient of 0.58). This work presents a sensitive proof-of-concept protocol for sputum liquefaction and decontamination followed by a simple DNA extraction procedure, in which the extraction steps are streamlined with a ready-to-use qPCR in a portable instrument that can be employed in low infrastructure settings.</description><subject>Bacilli</subject><subject>Biology and Life Sciences</subject><subject>Brazil</subject><subject>Cell culture</subject><subject>Costs</subject><subject>Culture</subject><subject>Decontamination</subject><subject>Deoxyribonucleic acid</subject><subject>Diagnosis</subject><subject>Dilution</subject><subject>DNA</subject><subject>DNA, Bacterial - isolation & purification</subject><subject>Filter paper</subject><subject>Genomics</subject><subject>Guanidine</subject><subject>Humans</subject><subject>Impurities</subject><subject>Isothiocyanate</subject><subject>Leprosy</subject><subject>Limit of Detection</subject><subject>Liquefaction</subject><subject>Mass Screening - economics</subject><subject>Mass Screening - instrumentation</subject><subject>Mass Screening - methods</subject><subject>Medical diagnosis</subject><subject>Medical screening</subject><subject>Medical tests</subject><subject>Medicine and Health Sciences</subject><subject>Methods</subject><subject>Microscopy</subject><subject>Mucin</subject><subject>Mycobacterium tuberculosis - genetics</subject><subject>Mycobacterium tuberculosis - isolation & purification</subject><subject>Nucleic acids</subject><subject>Nucleotide sequence</subject><subject>Physical Sciences</subject><subject>Point-of-Care Systems</subject><subject>Proof of Concept Study</subject><subject>Punches</subject><subject>Reagents</subject><subject>Real-Time Polymerase Chain Reaction - economics</subject><subject>Real-Time Polymerase Chain Reaction - instrumentation</subject><subject>Real-Time Polymerase Chain Reaction - methods</subject><subject>Research and analysis methods</subject><subject>Sample preparation</subject><subject>Sensitivity</subject><subject>Specimen Handling - economics</subject><subject>Specimen Handling - methods</subject><subject>Sputum</subject><subject>Sputum - microbiology</subject><subject>Tuberculosis</subject><subject>Tuberculosis, Pulmonary - diagnosis</subject><subject>Tuberculosis, Pulmonary - microbiology</subject><subject>Viscosity</subject><issn>1932-6203</issn><issn>1932-6203</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2020</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><sourceid>ABUWG</sourceid><sourceid>AFKRA</sourceid><sourceid>AZQEC</sourceid><sourceid>BENPR</sourceid><sourceid>CCPQU</sourceid><sourceid>DWQXO</sourceid><sourceid>GNUQQ</sourceid><sourceid>DOA</sourceid><recordid>eNqNk12L1DAUhoso7jr6D0QDguhFx3y0aeuFMKxfAwsLft2GTHo6k6FNapKq8xP816ZOd5nKXtgW2ibPeU_ynpwkeUzwkrCCvNrbwRnZLntrYIlpRjNc3knOScVoyilmd0--z5IH3u8xzlnJ-f3kjDFG8rLMz5Pfb6Gzxgcng7YG2QZJ1EgfkDQ1AukPyMuubyENNpXG_wSHemeDVbZFjXUoDBtwamit1x555QCMNlukDeqtNiG1TaqkA-QhhDjhX6PVKBDzxEdZo6APyIehPjxM7jWy9fBoei-Sr-_ffbn4mF5efVhfrC5TxSsaUmBl1jQKF0xWHFMCOS9pRZXiBLOSkCIjNWMVy0qucsYpFBUUXBGWKVAAnC2Sp0fdPi5aTC56QbMCY4LL6NYiWR-J2sq96J3upDsIK7X4O2DdVkgXtGpB5JuqzBtVRGGVYYCNpBsCBdQ5J_GfRK03U7Zh00GtwESr25nofMbondjaH6IoWM5jnRbJi0nA2e8D-CA67RW0rTRgh-O6aVHFK6LP_kFv391EbWXcgDaNjXnVKCpWPIsnhJFsTLu8hYp3DZ2OdYNGx_FZwMtZQGQC_ApbOXgv1p8__T979W3OPj9hdyDbsPO2Hcbj6udgdgSVs947aG5MJliMHXPthhg7RkwdE8OenBboJui6RdgfMPkRxg</recordid><startdate>20201214</startdate><enddate>20201214</enddate><creator>Ali, Nasir</creator><creator>Bello, Graziele Lima</creator><creator>Rossetti, Maria Lúcia Rosa</creator><creator>Krieger, Marco Aurelio</creator><creator>Costa, Alexandre Dias Tavares</creator><general>Public Library of Science</general><general>Public Library of Science (PLoS)</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>IOV</scope><scope>ISR</scope><scope>3V.</scope><scope>7QG</scope><scope>7QL</scope><scope>7QO</scope><scope>7RV</scope><scope>7SN</scope><scope>7SS</scope><scope>7T5</scope><scope>7TG</scope><scope>7TM</scope><scope>7U9</scope><scope>7X2</scope><scope>7X7</scope><scope>7XB</scope><scope>88E</scope><scope>8AO</scope><scope>8C1</scope><scope>8FD</scope><scope>8FE</scope><scope>8FG</scope><scope>8FH</scope><scope>8FI</scope><scope>8FJ</scope><scope>8FK</scope><scope>ABJCF</scope><scope>ABUWG</scope><scope>AEUYN</scope><scope>AFKRA</scope><scope>ARAPS</scope><scope>ATCPS</scope><scope>AZQEC</scope><scope>BBNVY</scope><scope>BENPR</scope><scope>BGLVJ</scope><scope>BHPHI</scope><scope>C1K</scope><scope>CCPQU</scope><scope>D1I</scope><scope>DWQXO</scope><scope>FR3</scope><scope>FYUFA</scope><scope>GHDGH</scope><scope>GNUQQ</scope><scope>H94</scope><scope>HCIFZ</scope><scope>K9.</scope><scope>KB.</scope><scope>KB0</scope><scope>KL.</scope><scope>L6V</scope><scope>LK8</scope><scope>M0K</scope><scope>M0S</scope><scope>M1P</scope><scope>M7N</scope><scope>M7P</scope><scope>M7S</scope><scope>NAPCQ</scope><scope>P5Z</scope><scope>P62</scope><scope>P64</scope><scope>PATMY</scope><scope>PDBOC</scope><scope>PIMPY</scope><scope>PQEST</scope><scope>PQQKQ</scope><scope>PQUKI</scope><scope>PTHSS</scope><scope>PYCSY</scope><scope>RC3</scope><scope>7X8</scope><scope>5PM</scope><scope>DOA</scope><orcidid>https://orcid.org/0000-0001-8434-2152</orcidid></search><sort><creationdate>20201214</creationdate><title>Demonstration of a fast and easy sample-to-answer protocol for tuberculosis screening in point-of-care settings: A proof of concept study</title><author>Ali, Nasir ; Bello, Graziele Lima ; Rossetti, Maria Lúcia Rosa ; Krieger, Marco Aurelio ; Costa, Alexandre Dias Tavares</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c692t-e384ffc073a96021e568292cc6103811741d3393486c5362e79e76c134cecee63</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2020</creationdate><topic>Bacilli</topic><topic>Biology and Life Sciences</topic><topic>Brazil</topic><topic>Cell culture</topic><topic>Costs</topic><topic>Culture</topic><topic>Decontamination</topic><topic>Deoxyribonucleic acid</topic><topic>Diagnosis</topic><topic>Dilution</topic><topic>DNA</topic><topic>DNA, Bacterial - isolation & purification</topic><topic>Filter paper</topic><topic>Genomics</topic><topic>Guanidine</topic><topic>Humans</topic><topic>Impurities</topic><topic>Isothiocyanate</topic><topic>Leprosy</topic><topic>Limit of Detection</topic><topic>Liquefaction</topic><topic>Mass Screening - economics</topic><topic>Mass Screening - instrumentation</topic><topic>Mass Screening - methods</topic><topic>Medical diagnosis</topic><topic>Medical screening</topic><topic>Medical tests</topic><topic>Medicine and Health Sciences</topic><topic>Methods</topic><topic>Microscopy</topic><topic>Mucin</topic><topic>Mycobacterium tuberculosis - genetics</topic><topic>Mycobacterium tuberculosis - isolation & purification</topic><topic>Nucleic acids</topic><topic>Nucleotide sequence</topic><topic>Physical Sciences</topic><topic>Point-of-Care Systems</topic><topic>Proof of Concept Study</topic><topic>Punches</topic><topic>Reagents</topic><topic>Real-Time Polymerase Chain Reaction - economics</topic><topic>Real-Time Polymerase Chain Reaction - 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Academic</collection><collection>PubMed Central (Full Participant titles)</collection><collection>DOAJ Directory of Open Access Journals</collection><jtitle>PloS one</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Ali, Nasir</au><au>Bello, Graziele Lima</au><au>Rossetti, Maria Lúcia Rosa</au><au>Krieger, Marco Aurelio</au><au>Costa, Alexandre Dias Tavares</au><au>Hasnain, Seyed Ehtesham</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Demonstration of a fast and easy sample-to-answer protocol for tuberculosis screening in point-of-care settings: A proof of concept study</atitle><jtitle>PloS one</jtitle><addtitle>PLoS One</addtitle><date>2020-12-14</date><risdate>2020</risdate><volume>15</volume><issue>12</issue><spage>e0242408</spage><epage>e0242408</epage><pages>e0242408-e0242408</pages><issn>1932-6203</issn><eissn>1932-6203</eissn><abstract>We sought to develop a smooth and low cost sample preparation and DNA extraction protocol, streamlined with a ready-to-use qPCR in a portable instrument to overcome some of the existing hurdles. Several solutions were evaluated as to their ability to liquefy a mucin-based matrix. Each liquefied matrix, supplemented with either Mycobacterium tuberculosis (MTB) H37Rv strain DNA or intact cells, was aliquoted onto a filter paper embedded with solubilizing agents, and was subsequently dried up. Most of the nucleic acids, including genomic DNA from the bacilli and the host, binds to the filter paper. Next, several protocols were evaluated to elute the DNA from the paper, using qPCR to detect the insertion sequence IS6110, a M. tuberculosis complex genomic marker. The limit of detection (LOD) of the best protocol was then evaluated using parallel seeding and colony counting. The protocol was also evaluated using seventeen sputum samples, previously characterized by the GeneXpert or culture. Two instruments (the ABI7500 Standard and the Q3-Plus system) and two reagents storage formats (frozen or ready-to-use) were evaluated. Solutions containing guanidine isothiocyanate exerted the best liquefying effect on the mucin-based matrix extracted from one 6-mm punches, followed by a brief incubation at 95°C. The resulting DNA contained impurities, but a simple 1:10 dilution elicited the detection of MTB and human genomic targets. The described protocol presented an apparent LOD of 02 CFU/mL of MTB. Challenging the protocol with previously characterized samples showed substantial agreement with GeneXpert MTB/RIF results (sensitivity of 90%, agreement of 88.9%, kappa coefficient of 0.77), and moderate agreement with culture results (sensitivity of 100%, agreement of 78.9%, kappa coefficient of 0.58). This work presents a sensitive proof-of-concept protocol for sputum liquefaction and decontamination followed by a simple DNA extraction procedure, in which the extraction steps are streamlined with a ready-to-use qPCR in a portable instrument that can be employed in low infrastructure settings.</abstract><cop>United States</cop><pub>Public Library of Science</pub><pmid>33315885</pmid><doi>10.1371/journal.pone.0242408</doi><tpages>e0242408</tpages><orcidid>https://orcid.org/0000-0001-8434-2152</orcidid><oa>free_for_read</oa></addata></record> |
| fulltext | fulltext |
| identifier | ISSN: 1932-6203 |
| ispartof | PloS one, 2020-12, Vol.15 (12), p.e0242408-e0242408 |
| issn | 1932-6203 1932-6203 |
| language | eng |
| recordid | cdi_plos_journals_2470010820 |
| source | MEDLINE; DOAJ Directory of Open Access Journals; Elektronische Zeitschriftenbibliothek - Frei zugängliche E-Journals; Public Library of Science (PLoS); PubMed Central; Free Full-Text Journals in Chemistry |
| subjects | Bacilli Biology and Life Sciences Brazil Cell culture Costs Culture Decontamination Deoxyribonucleic acid Diagnosis Dilution DNA DNA, Bacterial - isolation & purification Filter paper Genomics Guanidine Humans Impurities Isothiocyanate Leprosy Limit of Detection Liquefaction Mass Screening - economics Mass Screening - instrumentation Mass Screening - methods Medical diagnosis Medical screening Medical tests Medicine and Health Sciences Methods Microscopy Mucin Mycobacterium tuberculosis - genetics Mycobacterium tuberculosis - isolation & purification Nucleic acids Nucleotide sequence Physical Sciences Point-of-Care Systems Proof of Concept Study Punches Reagents Real-Time Polymerase Chain Reaction - economics Real-Time Polymerase Chain Reaction - instrumentation Real-Time Polymerase Chain Reaction - methods Research and analysis methods Sample preparation Sensitivity Specimen Handling - economics Specimen Handling - methods Sputum Sputum - microbiology Tuberculosis Tuberculosis, Pulmonary - diagnosis Tuberculosis, Pulmonary - microbiology Viscosity |
| title | Demonstration of a fast and easy sample-to-answer protocol for tuberculosis screening in point-of-care settings: A proof of concept study |
| url | https://sfx.bib-bvb.de/sfx_tum?ctx_ver=Z39.88-2004&ctx_enc=info:ofi/enc:UTF-8&ctx_tim=2025-01-20T10%3A03%3A49IST&url_ver=Z39.88-2004&url_ctx_fmt=infofi/fmt:kev:mtx:ctx&rfr_id=info:sid/primo.exlibrisgroup.com:primo3-Article-gale_plos_&rft_val_fmt=info:ofi/fmt:kev:mtx:journal&rft.genre=article&rft.atitle=Demonstration%20of%20a%20fast%20and%20easy%20sample-to-answer%20protocol%20for%20tuberculosis%20screening%20in%20point-of-care%20settings:%20A%20proof%20of%20concept%20study&rft.jtitle=PloS%20one&rft.au=Ali,%20Nasir&rft.date=2020-12-14&rft.volume=15&rft.issue=12&rft.spage=e0242408&rft.epage=e0242408&rft.pages=e0242408-e0242408&rft.issn=1932-6203&rft.eissn=1932-6203&rft_id=info:doi/10.1371/journal.pone.0242408&rft_dat=%3Cgale_plos_%3EA645383143%3C/gale_plos_%3E%3Curl%3E%3C/url%3E&disable_directlink=true&sfx.directlink=off&sfx.report_link=0&rft_id=info:oai/&rft_pqid=2470010820&rft_id=info:pmid/33315885&rft_galeid=A645383143&rft_doaj_id=oai_doaj_org_article_5b985fc7ee6c40eeba2b1e7ed56140e1&rfr_iscdi=true |