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Development of a Real-Time PCR Assay to Identify and Distinguish between Cryptococcus neoformans and Cryptococcus gattii Species Complexes
and are the principle causative agents of cryptococcosis. Differences in epidemiological and clinical features, and also treatment, mean it is important for diagnostic laboratories to distinguish between the two species. Molecular methods are potentially more rapid than culture and cryptococcal anti...
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| Published in: | Journal of fungi (Basel) 2022-04, Vol.8 (5), p.462 |
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| Main Authors: | , , , , |
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| Language: | English |
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| container_issue | 5 |
| container_start_page | 462 |
| container_title | Journal of fungi (Basel) |
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| creator | Tay, Enoch Chen, Sharon C-A Green, Wendy Lopez, Ronald Halliday, Catriona L |
| description | and
are the principle causative agents of cryptococcosis. Differences in epidemiological and clinical features, and also treatment, mean it is important for diagnostic laboratories to distinguish between the two species. Molecular methods are potentially more rapid than culture and cryptococcal antigen (CRAG) detection; however, commercial PCR-based assays that target
do not distinguish between species. Here, we developed a real-time PCR assay targeting the multicopy mitochondrial cytochrome
(
) gene to detect
and
in clinical specimens. Assay performance was compared with culture, histopathology, CRAG and panfungal PCR/DNA sequencing. The
-directed assay accurately detected and identified all eight
/
genotypes. High-resolution melt curve analysis unambiguously discriminated between the two species. Overall, assay sensitivity (96.4%) compared favorably with panfungal PCR (76.9%) and culture (14.5%); assay specificity was 100%. Of 25 fresh frozen paraffin embedded (FFPE) specimens, assay sensitivity was 96% (76% for panfungal PCR; 68% for histopathology). The
-specific PCR is a rapid (~4 h) sensitive method to diagnose (or exclude) cryptococcosis and differentiate between the two major species. It is suitable for use on diverse clinical specimens and may be the preferred molecular method for FFPE specimens where clinical suspicion of cryptococcosis is high. |
| doi_str_mv | 10.3390/jof8050462 |
| format | article |
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are the principle causative agents of cryptococcosis. Differences in epidemiological and clinical features, and also treatment, mean it is important for diagnostic laboratories to distinguish between the two species. Molecular methods are potentially more rapid than culture and cryptococcal antigen (CRAG) detection; however, commercial PCR-based assays that target
do not distinguish between species. Here, we developed a real-time PCR assay targeting the multicopy mitochondrial cytochrome
(
) gene to detect
and
in clinical specimens. Assay performance was compared with culture, histopathology, CRAG and panfungal PCR/DNA sequencing. The
-directed assay accurately detected and identified all eight
/
genotypes. High-resolution melt curve analysis unambiguously discriminated between the two species. Overall, assay sensitivity (96.4%) compared favorably with panfungal PCR (76.9%) and culture (14.5%); assay specificity was 100%. Of 25 fresh frozen paraffin embedded (FFPE) specimens, assay sensitivity was 96% (76% for panfungal PCR; 68% for histopathology). The
-specific PCR is a rapid (~4 h) sensitive method to diagnose (or exclude) cryptococcosis and differentiate between the two major species. It is suitable for use on diverse clinical specimens and may be the preferred molecular method for FFPE specimens where clinical suspicion of cryptococcosis is high.</description><identifier>ISSN: 2309-608X</identifier><identifier>EISSN: 2309-608X</identifier><identifier>DOI: 10.3390/jof8050462</identifier><identifier>PMID: 35628719</identifier><language>eng</language><publisher>Switzerland: MDPI AG</publisher><subject>Cryptococcosis ; Cryptococcus ; Cryptococcus gattii ; Cryptococcus neoformans ; Cryptococcus PCR ; Cytochrome ; Cytochrome b ; diagnosis of cryptococcosis ; DNA sequencing ; Epidemiology ; Genotypes ; Laboratories ; Microscopy ; Mitochondria ; Paraffin ; Pathogens ; Polymerase chain reaction ; Species</subject><ispartof>Journal of fungi (Basel), 2022-04, Vol.8 (5), p.462</ispartof><rights>2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/). Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License.</rights><rights>2022 by the authors. 2022</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c3872-5e43ca3fc3a39e90cacdbe68b10a4467b71fab7ef0d97ba40f521448b8e067103</citedby><cites>FETCH-LOGICAL-c3872-5e43ca3fc3a39e90cacdbe68b10a4467b71fab7ef0d97ba40f521448b8e067103</cites><orcidid>0000-0002-0301-7110</orcidid></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.proquest.com/docview/2670160032/fulltextPDF?pq-origsite=primo$$EPDF$$P50$$Gproquest$$Hfree_for_read</linktopdf><linktohtml>$$Uhttps://www.proquest.com/docview/2670160032?pq-origsite=primo$$EHTML$$P50$$Gproquest$$Hfree_for_read</linktohtml><link.rule.ids>230,314,727,780,784,885,25753,27924,27925,37012,37013,44590,53791,53793,75126</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/35628719$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Tay, Enoch</creatorcontrib><creatorcontrib>Chen, Sharon C-A</creatorcontrib><creatorcontrib>Green, Wendy</creatorcontrib><creatorcontrib>Lopez, Ronald</creatorcontrib><creatorcontrib>Halliday, Catriona L</creatorcontrib><title>Development of a Real-Time PCR Assay to Identify and Distinguish between Cryptococcus neoformans and Cryptococcus gattii Species Complexes</title><title>Journal of fungi (Basel)</title><addtitle>J Fungi (Basel)</addtitle><description>and
are the principle causative agents of cryptococcosis. Differences in epidemiological and clinical features, and also treatment, mean it is important for diagnostic laboratories to distinguish between the two species. Molecular methods are potentially more rapid than culture and cryptococcal antigen (CRAG) detection; however, commercial PCR-based assays that target
do not distinguish between species. Here, we developed a real-time PCR assay targeting the multicopy mitochondrial cytochrome
(
) gene to detect
and
in clinical specimens. Assay performance was compared with culture, histopathology, CRAG and panfungal PCR/DNA sequencing. The
-directed assay accurately detected and identified all eight
/
genotypes. High-resolution melt curve analysis unambiguously discriminated between the two species. Overall, assay sensitivity (96.4%) compared favorably with panfungal PCR (76.9%) and culture (14.5%); assay specificity was 100%. Of 25 fresh frozen paraffin embedded (FFPE) specimens, assay sensitivity was 96% (76% for panfungal PCR; 68% for histopathology). The
-specific PCR is a rapid (~4 h) sensitive method to diagnose (or exclude) cryptococcosis and differentiate between the two major species. It is suitable for use on diverse clinical specimens and may be the preferred molecular method for FFPE specimens where clinical suspicion of cryptococcosis is high.</description><subject>Cryptococcosis</subject><subject>Cryptococcus</subject><subject>Cryptococcus gattii</subject><subject>Cryptococcus neoformans</subject><subject>Cryptococcus PCR</subject><subject>Cytochrome</subject><subject>Cytochrome b</subject><subject>diagnosis of cryptococcosis</subject><subject>DNA sequencing</subject><subject>Epidemiology</subject><subject>Genotypes</subject><subject>Laboratories</subject><subject>Microscopy</subject><subject>Mitochondria</subject><subject>Paraffin</subject><subject>Pathogens</subject><subject>Polymerase chain reaction</subject><subject>Species</subject><issn>2309-608X</issn><issn>2309-608X</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2022</creationdate><recordtype>article</recordtype><sourceid>PIMPY</sourceid><sourceid>DOA</sourceid><recordid>eNpdkl9rFDEUxQdRbKl98QNIwBcRRm8mM5PkRShT_ywUlFrBt5DJ3GyzzEzGZKa6X8FPbbZba1dCyCX3x-FwOFn2nMIbxiS83XgroIKyLh5lxwUDmdcgvj9-MB9lpzFuAIBWopaSPc2OWFUXglN5nP0-xxvs_TTgOBNviSaXqPv8yg1IvjSX5CxGvSWzJ6suEc5uiR47cu7i7Mb14uI1aXH-iTiSJmyn2RtvzBLJiN76MOgx3vIHu7WeZ-fI1wmNw0gaP0w9_sL4LHtidR_x9O49yb59eH_VfMovPn9cNWcXuWGCF3mFJTOaWcM0kyjBaNO1WIuWgi7LmrecWt1ytNBJ3uoSbFXQshStQKg5BXaSrfa6ndcbNQU36LBVXjt1--HDWukwO9OjopU0tGqFpqYubauFTAc63lGD0BUiab3ba01LO2BnUkZB9weih5vRXau1v1EyWQLOk8CrO4HgfywYZzW4aLDvdYpwiapIltPl5Q59-R-68UsYU1Q7CmgNwIpEvd5TJvgYA9p7MxTUrjHqX2MS_OKh_Xv0bz_YHw8nvjQ</recordid><startdate>20220429</startdate><enddate>20220429</enddate><creator>Tay, Enoch</creator><creator>Chen, Sharon C-A</creator><creator>Green, Wendy</creator><creator>Lopez, Ronald</creator><creator>Halliday, Catriona L</creator><general>MDPI AG</general><general>MDPI</general><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>8FE</scope><scope>8FH</scope><scope>ABUWG</scope><scope>AFKRA</scope><scope>AZQEC</scope><scope>BBNVY</scope><scope>BENPR</scope><scope>BHPHI</scope><scope>CCPQU</scope><scope>DWQXO</scope><scope>GNUQQ</scope><scope>HCIFZ</scope><scope>LK8</scope><scope>M7P</scope><scope>PIMPY</scope><scope>PQEST</scope><scope>PQQKQ</scope><scope>PQUKI</scope><scope>PRINS</scope><scope>7X8</scope><scope>5PM</scope><scope>DOA</scope><orcidid>https://orcid.org/0000-0002-0301-7110</orcidid></search><sort><creationdate>20220429</creationdate><title>Development of a Real-Time PCR Assay to Identify and Distinguish between Cryptococcus neoformans and Cryptococcus gattii Species Complexes</title><author>Tay, Enoch ; Chen, Sharon C-A ; Green, Wendy ; Lopez, Ronald ; Halliday, Catriona L</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c3872-5e43ca3fc3a39e90cacdbe68b10a4467b71fab7ef0d97ba40f521448b8e067103</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2022</creationdate><topic>Cryptococcosis</topic><topic>Cryptococcus</topic><topic>Cryptococcus gattii</topic><topic>Cryptococcus neoformans</topic><topic>Cryptococcus PCR</topic><topic>Cytochrome</topic><topic>Cytochrome b</topic><topic>diagnosis of cryptococcosis</topic><topic>DNA sequencing</topic><topic>Epidemiology</topic><topic>Genotypes</topic><topic>Laboratories</topic><topic>Microscopy</topic><topic>Mitochondria</topic><topic>Paraffin</topic><topic>Pathogens</topic><topic>Polymerase chain reaction</topic><topic>Species</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Tay, Enoch</creatorcontrib><creatorcontrib>Chen, Sharon C-A</creatorcontrib><creatorcontrib>Green, Wendy</creatorcontrib><creatorcontrib>Lopez, Ronald</creatorcontrib><creatorcontrib>Halliday, Catriona L</creatorcontrib><collection>PubMed</collection><collection>CrossRef</collection><collection>ProQuest SciTech Collection</collection><collection>ProQuest Natural Science Collection</collection><collection>ProQuest Central (Alumni)</collection><collection>ProQuest Central</collection><collection>ProQuest Central Essentials</collection><collection>Biological Science Collection</collection><collection>ProQuest Central</collection><collection>ProQuest Natural Science Collection</collection><collection>ProQuest One Community College</collection><collection>ProQuest Central</collection><collection>ProQuest Central Student</collection><collection>SciTech Premium Collection</collection><collection>ProQuest Biological Science Collection</collection><collection>ProQuest Biological Science Journals</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>ProQuest Central China</collection><collection>MEDLINE - Academic</collection><collection>PubMed Central (Full Participant titles)</collection><collection>Open Access: DOAJ - Directory of Open Access Journals</collection><jtitle>Journal of fungi (Basel)</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Tay, Enoch</au><au>Chen, Sharon C-A</au><au>Green, Wendy</au><au>Lopez, Ronald</au><au>Halliday, Catriona L</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Development of a Real-Time PCR Assay to Identify and Distinguish between Cryptococcus neoformans and Cryptococcus gattii Species Complexes</atitle><jtitle>Journal of fungi (Basel)</jtitle><addtitle>J Fungi (Basel)</addtitle><date>2022-04-29</date><risdate>2022</risdate><volume>8</volume><issue>5</issue><spage>462</spage><pages>462-</pages><issn>2309-608X</issn><eissn>2309-608X</eissn><abstract>and
are the principle causative agents of cryptococcosis. Differences in epidemiological and clinical features, and also treatment, mean it is important for diagnostic laboratories to distinguish between the two species. Molecular methods are potentially more rapid than culture and cryptococcal antigen (CRAG) detection; however, commercial PCR-based assays that target
do not distinguish between species. Here, we developed a real-time PCR assay targeting the multicopy mitochondrial cytochrome
(
) gene to detect
and
in clinical specimens. Assay performance was compared with culture, histopathology, CRAG and panfungal PCR/DNA sequencing. The
-directed assay accurately detected and identified all eight
/
genotypes. High-resolution melt curve analysis unambiguously discriminated between the two species. Overall, assay sensitivity (96.4%) compared favorably with panfungal PCR (76.9%) and culture (14.5%); assay specificity was 100%. Of 25 fresh frozen paraffin embedded (FFPE) specimens, assay sensitivity was 96% (76% for panfungal PCR; 68% for histopathology). The
-specific PCR is a rapid (~4 h) sensitive method to diagnose (or exclude) cryptococcosis and differentiate between the two major species. It is suitable for use on diverse clinical specimens and may be the preferred molecular method for FFPE specimens where clinical suspicion of cryptococcosis is high.</abstract><cop>Switzerland</cop><pub>MDPI AG</pub><pmid>35628719</pmid><doi>10.3390/jof8050462</doi><orcidid>https://orcid.org/0000-0002-0301-7110</orcidid><oa>free_for_read</oa></addata></record> |
| fulltext | fulltext |
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| ispartof | Journal of fungi (Basel), 2022-04, Vol.8 (5), p.462 |
| issn | 2309-608X 2309-608X |
| language | eng |
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| source | Open Access: PubMed Central; Publicly Available Content Database |
| subjects | Cryptococcosis Cryptococcus Cryptococcus gattii Cryptococcus neoformans Cryptococcus PCR Cytochrome Cytochrome b diagnosis of cryptococcosis DNA sequencing Epidemiology Genotypes Laboratories Microscopy Mitochondria Paraffin Pathogens Polymerase chain reaction Species |
| title | Development of a Real-Time PCR Assay to Identify and Distinguish between Cryptococcus neoformans and Cryptococcus gattii Species Complexes |
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