Development of a novel real-time polymerase chain reaction assay for the sensitive detection of Schistosoma japonicum in human stool
Elimination and control of Schistosoma japonicum, the most virulent of the schistosomiasis-causing blood flukes, requires the development of sensitive and specific diagnostic tools capable of providing an accurate measurement of the infection prevalence in endemic areas. Typically, detection of S. j...
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| description | Elimination and control of Schistosoma japonicum, the most virulent of the schistosomiasis-causing blood flukes, requires the development of sensitive and specific diagnostic tools capable of providing an accurate measurement of the infection prevalence in endemic areas. Typically, detection of S. japonicum has occurred using the Kato-Katz technique, but this methodology, which requires skilled microscopists, has been shown to radically underestimate levels of infection. With the ever-improving capabilities of next-generation sequencing and bioinformatic analysis tools, identification of satellite sequences and other highly repetitive genomic elements for use as real-time PCR diagnostic targets is becoming increasingly common. Assays developed using these targets have the ability to improve the sensitivity and specificity of results for epidemiological studies that can in turn be used to inform mass drug administration and programmatic decision making.
Utilizing Tandem Repeat Analyzer (TAREAN) and RepeatExplorer2, a cluster-based analysis of the S. japonicum genome was performed and a tandemly arranged genomic repeat, which we named SjTR1 (Schistosoma japonicum Tandem Repeat 1), was selected as the target for a real-time PCR diagnostic assay. Based on these analyses, a primer/probe set was designed and the assay was optimized. The resulting real-time PCR test was shown to reliably detect as little as 200 ag of S. japonicum genomic DNA and as little as 1 egg per gram of human stool. Based on these results, the index assay reported in this manuscript is more sensitive than previously published real-time PCR assays for the detection of S. japonicum.
The extremely sensitive and specific diagnostic assay described in this manuscript will facilitate the accurate detection of S. japonicum, particularly in regions with low levels of endemicity. This assay will be useful in providing data to inform programmatic decision makers, aiding disease control and elimination efforts. |
| doi_str_mv | 10.1371/journal.pntd.0009877 |
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Utilizing Tandem Repeat Analyzer (TAREAN) and RepeatExplorer2, a cluster-based analysis of the S. japonicum genome was performed and a tandemly arranged genomic repeat, which we named SjTR1 (Schistosoma japonicum Tandem Repeat 1), was selected as the target for a real-time PCR diagnostic assay. Based on these analyses, a primer/probe set was designed and the assay was optimized. The resulting real-time PCR test was shown to reliably detect as little as 200 ag of S. japonicum genomic DNA and as little as 1 egg per gram of human stool. Based on these results, the index assay reported in this manuscript is more sensitive than previously published real-time PCR assays for the detection of S. japonicum.
The extremely sensitive and specific diagnostic assay described in this manuscript will facilitate the accurate detection of S. japonicum, particularly in regions with low levels of endemicity. This assay will be useful in providing data to inform programmatic decision makers, aiding disease control and elimination efforts.</description><identifier>ISSN: 1935-2735</identifier><identifier>ISSN: 1935-2727</identifier><identifier>EISSN: 1935-2735</identifier><identifier>DOI: 10.1371/journal.pntd.0009877</identifier><identifier>PMID: 34695134</identifier><language>eng</language><publisher>United States: Public Library of Science</publisher><subject>Animals ; Annealing ; Assaying ; Binding sites ; Biochemical assays ; Biology and Life Sciences ; Decision analysis ; Decision making ; Deoxyribonucleic acid ; Detection ; Diagnosis ; Disease control ; DNA ; DNA Primers - genetics ; Efficiency ; Eggs ; Endemism ; Epidemiology ; Ethics ; Feces - parasitology ; Female ; Genomes ; Genomics ; Human wastes ; Humans ; Infections ; Male ; Methods ; Microscopy ; Mitochondrial DNA ; Molecular diagnostic techniques ; Next-generation sequencing ; Nucleotide sequence ; PCR ; Polymerase chain reaction ; Real time ; Real-Time Polymerase Chain Reaction - methods ; Research and Analysis Methods ; Schistosoma japonicum ; Schistosoma japonicum - genetics ; Schistosoma japonicum - isolation & purification ; Schistosomiasis ; Schistosomiasis japonica - diagnosis ; Schistosomiasis japonica - parasitology ; Sensitivity and Specificity ; Specificity ; Tropical diseases</subject><ispartof>PLoS neglected tropical diseases, 2021-10, Vol.15 (10), p.e0009877-e0009877</ispartof><rights>COPYRIGHT 2021 Public Library of Science</rights><rights>2021 Halili 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>2021 Halili et al 2021 Halili et al</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c624t-9d2f0bdc7d9f5cfab44237cef418b31e4c11b225b14aa348c8feb22bd85a19a03</citedby><cites>FETCH-LOGICAL-c624t-9d2f0bdc7d9f5cfab44237cef418b31e4c11b225b14aa348c8feb22bd85a19a03</cites><orcidid>0000-0002-8447-7425 ; 0000-0002-1509-5220 ; 0000-0003-0586-8573 ; 0000-0002-0725-202X ; 0000-0002-4881-7496</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/PMC8568117/pdf/$$EPDF$$P50$$Gpubmedcentral$$Hfree_for_read</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC8568117/$$EHTML$$P50$$Gpubmedcentral$$Hfree_for_read</linktohtml><link.rule.ids>230,314,727,780,784,864,885,2100,2919,23857,27915,27916,53782,53784,79361,79362</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/34695134$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><contributor>Cantacessi, Cinzia</contributor><creatorcontrib>Halili, Sara</creatorcontrib><creatorcontrib>Grant, Jessica R</creatorcontrib><creatorcontrib>Pilotte, Nils</creatorcontrib><creatorcontrib>Gordon, Catherine A</creatorcontrib><creatorcontrib>Williams, Steven A</creatorcontrib><title>Development of a novel real-time polymerase chain reaction assay for the sensitive detection of Schistosoma japonicum in human stool</title><title>PLoS neglected tropical diseases</title><addtitle>PLoS Negl Trop Dis</addtitle><description>Elimination and control of Schistosoma japonicum, the most virulent of the schistosomiasis-causing blood flukes, requires the development of sensitive and specific diagnostic tools capable of providing an accurate measurement of the infection prevalence in endemic areas. Typically, detection of S. japonicum has occurred using the Kato-Katz technique, but this methodology, which requires skilled microscopists, has been shown to radically underestimate levels of infection. With the ever-improving capabilities of next-generation sequencing and bioinformatic analysis tools, identification of satellite sequences and other highly repetitive genomic elements for use as real-time PCR diagnostic targets is becoming increasingly common. Assays developed using these targets have the ability to improve the sensitivity and specificity of results for epidemiological studies that can in turn be used to inform mass drug administration and programmatic decision making.
Utilizing Tandem Repeat Analyzer (TAREAN) and RepeatExplorer2, a cluster-based analysis of the S. japonicum genome was performed and a tandemly arranged genomic repeat, which we named SjTR1 (Schistosoma japonicum Tandem Repeat 1), was selected as the target for a real-time PCR diagnostic assay. Based on these analyses, a primer/probe set was designed and the assay was optimized. The resulting real-time PCR test was shown to reliably detect as little as 200 ag of S. japonicum genomic DNA and as little as 1 egg per gram of human stool. Based on these results, the index assay reported in this manuscript is more sensitive than previously published real-time PCR assays for the detection of S. japonicum.
The extremely sensitive and specific diagnostic assay described in this manuscript will facilitate the accurate detection of S. japonicum, particularly in regions with low levels of endemicity. This assay will be useful in providing data to inform programmatic decision makers, aiding disease control and elimination efforts.</description><subject>Animals</subject><subject>Annealing</subject><subject>Assaying</subject><subject>Binding sites</subject><subject>Biochemical assays</subject><subject>Biology and Life Sciences</subject><subject>Decision analysis</subject><subject>Decision making</subject><subject>Deoxyribonucleic acid</subject><subject>Detection</subject><subject>Diagnosis</subject><subject>Disease control</subject><subject>DNA</subject><subject>DNA Primers - genetics</subject><subject>Efficiency</subject><subject>Eggs</subject><subject>Endemism</subject><subject>Epidemiology</subject><subject>Ethics</subject><subject>Feces - parasitology</subject><subject>Female</subject><subject>Genomes</subject><subject>Genomics</subject><subject>Human wastes</subject><subject>Humans</subject><subject>Infections</subject><subject>Male</subject><subject>Methods</subject><subject>Microscopy</subject><subject>Mitochondrial DNA</subject><subject>Molecular diagnostic techniques</subject><subject>Next-generation sequencing</subject><subject>Nucleotide sequence</subject><subject>PCR</subject><subject>Polymerase chain reaction</subject><subject>Real time</subject><subject>Real-Time Polymerase Chain Reaction - methods</subject><subject>Research and Analysis Methods</subject><subject>Schistosoma japonicum</subject><subject>Schistosoma japonicum - genetics</subject><subject>Schistosoma japonicum - isolation & purification</subject><subject>Schistosomiasis</subject><subject>Schistosomiasis japonica - diagnosis</subject><subject>Schistosomiasis japonica - parasitology</subject><subject>Sensitivity and Specificity</subject><subject>Specificity</subject><subject>Tropical diseases</subject><issn>1935-2735</issn><issn>1935-2727</issn><issn>1935-2735</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2021</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>DOA</sourceid><recordid>eNptkk1r3DAQhk1padK0_6C0gkLpZbeSJdvyJRDSr0Cgh7ZnMfqKtcjS1rIX9t4fXrnrhN0SdLA888w7o-EtitcErwltyMdNnIYAfr0No15jjFveNE-Kc9LSalU2tHp6dD8rXqS0wbhqK06eF2eU1W1FKDsv_nwyO-PjtjdhRNEiQCHmABoM-NXoeoO20e97M0AySHXgwpxSo4sBQUqwRzYOaOwMSiYkN7qdQdqM5kBkwR-qc2mMKfaANrCNwampR1mmm3oIKKeif1k8s-CTebV8L4pfXz7_vP62uv3-9eb66nal6pKNq1aXFkutGt3aSlmQjJW0UcYywiUlhilCZFlWkjAAyrji1uR_qXkFpAVML4q3B92tj0ksC0yirFpOcI2bmbg5EDrCRmwH18OwFxGc-BeIw52AYXTKGwFay0pjbK1UjGoquW0pYKtKLRstSda6XLpNsjda5Q0P4E9ETzPBdeIu7gSvak5IkwU-LAJD_D2ZNIreJWW8h2DiNM_Na8baumYZffcf-vjrFuoO8gNcsDH3VbOouKp5SdrcdJ57_QiVjza9UzEY63L8pOD9UUGXnTN2KfpptkA6BdkBVENMaTD2YRkEi9nU91OL2dRiMXUue3O8yIeiexfTv5Gb968</recordid><startdate>20211001</startdate><enddate>20211001</enddate><creator>Halili, Sara</creator><creator>Grant, Jessica R</creator><creator>Pilotte, Nils</creator><creator>Gordon, Catherine A</creator><creator>Williams, Steven A</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>3V.</scope><scope>7QL</scope><scope>7SS</scope><scope>7T2</scope><scope>7T7</scope><scope>7U9</scope><scope>7X7</scope><scope>7XB</scope><scope>88E</scope><scope>8C1</scope><scope>8FD</scope><scope>8FI</scope><scope>8FJ</scope><scope>8FK</scope><scope>ABUWG</scope><scope>AEUYN</scope><scope>AFKRA</scope><scope>AZQEC</scope><scope>BENPR</scope><scope>C1K</scope><scope>CCPQU</scope><scope>DWQXO</scope><scope>F1W</scope><scope>FR3</scope><scope>FYUFA</scope><scope>GHDGH</scope><scope>H94</scope><scope>H95</scope><scope>H97</scope><scope>K9.</scope><scope>L.G</scope><scope>M0S</scope><scope>M1P</scope><scope>M7N</scope><scope>P64</scope><scope>PIMPY</scope><scope>PQEST</scope><scope>PQQKQ</scope><scope>PQUKI</scope><scope>7X8</scope><scope>5PM</scope><scope>DOA</scope><orcidid>https://orcid.org/0000-0002-8447-7425</orcidid><orcidid>https://orcid.org/0000-0002-1509-5220</orcidid><orcidid>https://orcid.org/0000-0003-0586-8573</orcidid><orcidid>https://orcid.org/0000-0002-0725-202X</orcidid><orcidid>https://orcid.org/0000-0002-4881-7496</orcidid></search><sort><creationdate>20211001</creationdate><title>Development of a novel real-time polymerase chain reaction assay for the sensitive detection of Schistosoma japonicum in human stool</title><author>Halili, Sara ; Grant, Jessica R ; Pilotte, Nils ; Gordon, Catherine A ; Williams, Steven A</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c624t-9d2f0bdc7d9f5cfab44237cef418b31e4c11b225b14aa348c8feb22bd85a19a03</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2021</creationdate><topic>Animals</topic><topic>Annealing</topic><topic>Assaying</topic><topic>Binding sites</topic><topic>Biochemical assays</topic><topic>Biology and Life Sciences</topic><topic>Decision analysis</topic><topic>Decision making</topic><topic>Deoxyribonucleic acid</topic><topic>Detection</topic><topic>Diagnosis</topic><topic>Disease control</topic><topic>DNA</topic><topic>DNA Primers - genetics</topic><topic>Efficiency</topic><topic>Eggs</topic><topic>Endemism</topic><topic>Epidemiology</topic><topic>Ethics</topic><topic>Feces - parasitology</topic><topic>Female</topic><topic>Genomes</topic><topic>Genomics</topic><topic>Human wastes</topic><topic>Humans</topic><topic>Infections</topic><topic>Male</topic><topic>Methods</topic><topic>Microscopy</topic><topic>Mitochondrial DNA</topic><topic>Molecular diagnostic techniques</topic><topic>Next-generation sequencing</topic><topic>Nucleotide sequence</topic><topic>PCR</topic><topic>Polymerase chain reaction</topic><topic>Real time</topic><topic>Real-Time Polymerase Chain Reaction - methods</topic><topic>Research and Analysis Methods</topic><topic>Schistosoma japonicum</topic><topic>Schistosoma japonicum - genetics</topic><topic>Schistosoma japonicum - isolation & purification</topic><topic>Schistosomiasis</topic><topic>Schistosomiasis japonica - diagnosis</topic><topic>Schistosomiasis japonica - parasitology</topic><topic>Sensitivity and Specificity</topic><topic>Specificity</topic><topic>Tropical diseases</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Halili, Sara</creatorcontrib><creatorcontrib>Grant, Jessica R</creatorcontrib><creatorcontrib>Pilotte, Nils</creatorcontrib><creatorcontrib>Gordon, Catherine A</creatorcontrib><creatorcontrib>Williams, Steven A</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>ProQuest Central (Corporate)</collection><collection>Bacteriology Abstracts (Microbiology B)</collection><collection>Entomology Abstracts (Full archive)</collection><collection>Health and Safety Science Abstracts (Full archive)</collection><collection>Industrial and Applied Microbiology Abstracts (Microbiology A)</collection><collection>Virology and AIDS Abstracts</collection><collection>ProQuest Health & Medical Collection</collection><collection>ProQuest Central (purchase pre-March 2016)</collection><collection>Medical Database (Alumni Edition)</collection><collection>ProQuest Public Health Database</collection><collection>Technology Research Database</collection><collection>Hospital Premium Collection</collection><collection>Hospital Premium Collection (Alumni Edition)</collection><collection>ProQuest Central (Alumni) (purchase pre-March 2016)</collection><collection>ProQuest Central (Alumni)</collection><collection>ProQuest One Sustainability</collection><collection>ProQuest Central</collection><collection>ProQuest Central Essentials</collection><collection>ProQuest Central</collection><collection>Environmental Sciences and Pollution Management</collection><collection>ProQuest One Community College</collection><collection>ProQuest Central</collection><collection>ASFA: Aquatic Sciences and Fisheries Abstracts</collection><collection>Engineering Research Database</collection><collection>Health Research Premium Collection</collection><collection>Health Research Premium Collection (Alumni)</collection><collection>AIDS and Cancer Research Abstracts</collection><collection>Aquatic Science & Fisheries Abstracts (ASFA) 1: Biological Sciences & Living Resources</collection><collection>Aquatic Science & Fisheries Abstracts (ASFA) 3: Aquatic Pollution & Environmental Quality</collection><collection>ProQuest Health & Medical Complete (Alumni)</collection><collection>Aquatic Science & Fisheries Abstracts (ASFA) Professional</collection><collection>Health & Medical Collection (Alumni Edition)</collection><collection>PML(ProQuest Medical Library)</collection><collection>Algology Mycology and Protozoology Abstracts (Microbiology C)</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>Publicly Available Content Database</collection><collection>ProQuest One Academic Eastern Edition (DO NOT USE)</collection><collection>ProQuest One Academic</collection><collection>ProQuest One Academic UKI Edition</collection><collection>MEDLINE - Academic</collection><collection>PubMed Central (Full Participant titles)</collection><collection>Directory of Open Access Journals</collection><jtitle>PLoS neglected tropical diseases</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Halili, Sara</au><au>Grant, Jessica R</au><au>Pilotte, Nils</au><au>Gordon, Catherine A</au><au>Williams, Steven A</au><au>Cantacessi, Cinzia</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Development of a novel real-time polymerase chain reaction assay for the sensitive detection of Schistosoma japonicum in human stool</atitle><jtitle>PLoS neglected tropical diseases</jtitle><addtitle>PLoS Negl Trop Dis</addtitle><date>2021-10-01</date><risdate>2021</risdate><volume>15</volume><issue>10</issue><spage>e0009877</spage><epage>e0009877</epage><pages>e0009877-e0009877</pages><issn>1935-2735</issn><issn>1935-2727</issn><eissn>1935-2735</eissn><abstract>Elimination and control of Schistosoma japonicum, the most virulent of the schistosomiasis-causing blood flukes, requires the development of sensitive and specific diagnostic tools capable of providing an accurate measurement of the infection prevalence in endemic areas. Typically, detection of S. japonicum has occurred using the Kato-Katz technique, but this methodology, which requires skilled microscopists, has been shown to radically underestimate levels of infection. With the ever-improving capabilities of next-generation sequencing and bioinformatic analysis tools, identification of satellite sequences and other highly repetitive genomic elements for use as real-time PCR diagnostic targets is becoming increasingly common. Assays developed using these targets have the ability to improve the sensitivity and specificity of results for epidemiological studies that can in turn be used to inform mass drug administration and programmatic decision making.
Utilizing Tandem Repeat Analyzer (TAREAN) and RepeatExplorer2, a cluster-based analysis of the S. japonicum genome was performed and a tandemly arranged genomic repeat, which we named SjTR1 (Schistosoma japonicum Tandem Repeat 1), was selected as the target for a real-time PCR diagnostic assay. Based on these analyses, a primer/probe set was designed and the assay was optimized. The resulting real-time PCR test was shown to reliably detect as little as 200 ag of S. japonicum genomic DNA and as little as 1 egg per gram of human stool. Based on these results, the index assay reported in this manuscript is more sensitive than previously published real-time PCR assays for the detection of S. japonicum.
The extremely sensitive and specific diagnostic assay described in this manuscript will facilitate the accurate detection of S. japonicum, particularly in regions with low levels of endemicity. This assay will be useful in providing data to inform programmatic decision makers, aiding disease control and elimination efforts.</abstract><cop>United States</cop><pub>Public Library of Science</pub><pmid>34695134</pmid><doi>10.1371/journal.pntd.0009877</doi><orcidid>https://orcid.org/0000-0002-8447-7425</orcidid><orcidid>https://orcid.org/0000-0002-1509-5220</orcidid><orcidid>https://orcid.org/0000-0003-0586-8573</orcidid><orcidid>https://orcid.org/0000-0002-0725-202X</orcidid><orcidid>https://orcid.org/0000-0002-4881-7496</orcidid><oa>free_for_read</oa></addata></record> |
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| subjects | Animals Annealing Assaying Binding sites Biochemical assays Biology and Life Sciences Decision analysis Decision making Deoxyribonucleic acid Detection Diagnosis Disease control DNA DNA Primers - genetics Efficiency Eggs Endemism Epidemiology Ethics Feces - parasitology Female Genomes Genomics Human wastes Humans Infections Male Methods Microscopy Mitochondrial DNA Molecular diagnostic techniques Next-generation sequencing Nucleotide sequence PCR Polymerase chain reaction Real time Real-Time Polymerase Chain Reaction - methods Research and Analysis Methods Schistosoma japonicum Schistosoma japonicum - genetics Schistosoma japonicum - isolation & purification Schistosomiasis Schistosomiasis japonica - diagnosis Schistosomiasis japonica - parasitology Sensitivity and Specificity Specificity Tropical diseases |
| title | Development of a novel real-time polymerase chain reaction assay for the sensitive detection of Schistosoma japonicum in human stool |
| url | https://sfx.bib-bvb.de/sfx_tum?ctx_ver=Z39.88-2004&ctx_enc=info:ofi/enc:UTF-8&ctx_tim=2025-01-14T19%3A43%3A23IST&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=Development%20of%20a%20novel%20real-time%20polymerase%20chain%20reaction%20assay%20for%20the%20sensitive%20detection%20of%20Schistosoma%20japonicum%20in%20human%20stool&rft.jtitle=PLoS%20neglected%20tropical%20diseases&rft.au=Halili,%20Sara&rft.date=2021-10-01&rft.volume=15&rft.issue=10&rft.spage=e0009877&rft.epage=e0009877&rft.pages=e0009877-e0009877&rft.issn=1935-2735&rft.eissn=1935-2735&rft_id=info:doi/10.1371/journal.pntd.0009877&rft_dat=%3Cgale_plos_%3EA682191731%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=2598106070&rft_id=info:pmid/34695134&rft_galeid=A682191731&rft_doaj_id=oai_doaj_org_article_addb5d00ffbc43d3b8f93a0fc2db7db1&rfr_iscdi=true |