Comparison of quantitative real-time PCR and direct immunofluorescence for the detection of Pneumocystis jirovecii
Pneumocystis pneumonia (PCP) is a serious risk for HIV-positive patients. Asymptomatic infection or colonisation with P. jirovecii has been shown to occur frequently. PCR assays frequently identify such cases, due to their high sensitivity. Quantitative real-time PCR (qPCR) gene copy number cut-off...
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| description | Pneumocystis pneumonia (PCP) is a serious risk for HIV-positive patients. Asymptomatic infection or colonisation with P. jirovecii has been shown to occur frequently. PCR assays frequently identify such cases, due to their high sensitivity. Quantitative real-time PCR (qPCR) gene copy number cut-off values have been suggested to differentiate colonisation and infection; these need to be standardised for routine use. We compared the results of qPCR with an immunofluorescence assay (IFA) to determine a specific cut-off value.
From March 2005 through June 2009, induced sputum specimens were collected from adult patients who were clinically suspected of having PCP, at the Chris Hani Baragwanath Hospital in Gauteng, South Africa. Laboratory diagnosis of PCP was done by a conventional direct IFA and a qPCR assay. A receiver operating characteristic (ROC) analysis was performed to determine a suitable copy number cut-off value.
P. jirovecii was identified in 51% (156/305) and 67% (204/305) of specimens using IFA and qPCR, respectively. The cut-off value for the qPCR that best predicted the IFA results was 78 copies/5 μl (area under ROC curve 0.92). The sensitivity and specificity of qPCR using this cut-off was 94.6% and 89.1%, respectively, compared with the IFA.
The results of the ROC curve analysis indicate an excellent predictive value of the qPCR using the proposed cut-off. However, the IFA test is an imperfect gold standard and so this cut-off should not be used in isolation; clinical data should also contribute to the interpretation of the qPCR result. |
| doi_str_mv | 10.1371/journal.pone.0180589 |
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From March 2005 through June 2009, induced sputum specimens were collected from adult patients who were clinically suspected of having PCP, at the Chris Hani Baragwanath Hospital in Gauteng, South Africa. Laboratory diagnosis of PCP was done by a conventional direct IFA and a qPCR assay. A receiver operating characteristic (ROC) analysis was performed to determine a suitable copy number cut-off value.
P. jirovecii was identified in 51% (156/305) and 67% (204/305) of specimens using IFA and qPCR, respectively. The cut-off value for the qPCR that best predicted the IFA results was 78 copies/5 μl (area under ROC curve 0.92). The sensitivity and specificity of qPCR using this cut-off was 94.6% and 89.1%, respectively, compared with the IFA.
The results of the ROC curve analysis indicate an excellent predictive value of the qPCR using the proposed cut-off. However, the IFA test is an imperfect gold standard and so this cut-off should not be used in isolation; clinical data should also contribute to the interpretation of the qPCR result.</description><identifier>ISSN: 1932-6203</identifier><identifier>EISSN: 1932-6203</identifier><identifier>DOI: 10.1371/journal.pone.0180589</identifier><identifier>PMID: 28683092</identifier><language>eng</language><publisher>United States: Public Library of Science</publisher><subject>Adult ; Analysis ; Assaying ; Asymptomatic infection ; Biology and Life Sciences ; Colonization ; Copy number ; Cysts ; Deoxyribonucleic acid ; Diagnosis ; Disease control ; DNA ; Female ; Fluorescent antibody technique ; Fluorescent Antibody Technique, Direct - methods ; Health aspects ; Health risks ; HIV ; Human immunodeficiency virus ; Humans ; Immunofluorescence ; Infections ; Influenza ; Laboratories ; Male ; Medicine and Health Sciences ; Parasitic diseases ; Patients ; People and places ; Pneumocystis carinii - genetics ; Pneumocystis carinii - isolation & purification ; Pneumonia ; Pneumonia, Pneumocystis - diagnosis ; Pneumonia, Pneumocystis - microbiology ; Polymerase chain reaction ; Real time ; Real-Time Polymerase Chain Reaction - methods ; Research and Analysis Methods ; Sensitivity ; Sputum ; Zoonoses</subject><ispartof>PloS one, 2017-07, Vol.12 (7), p.e0180589-e0180589</ispartof><rights>COPYRIGHT 2017 Public Library of Science</rights><rights>2017 Moodley 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>2017 Moodley et al 2017 Moodley et al</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c692t-3dd1b9b68dd096a8e28fc7fc19aa4ff7cce160bcfe9b00d36724e9d88a0ec3693</citedby><cites>FETCH-LOGICAL-c692t-3dd1b9b68dd096a8e28fc7fc19aa4ff7cce160bcfe9b00d36724e9d88a0ec3693</cites><orcidid>0000-0002-2344-3811</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/PMC5500343/pdf/$$EPDF$$P50$$Gpubmedcentral$$Hfree_for_read</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC5500343/$$EHTML$$P50$$Gpubmedcentral$$Hfree_for_read</linktohtml><link.rule.ids>230,314,723,776,780,860,881,2096,2915,23845,27901,27902,53766,53768,79343,79344</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/28683092$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><contributor>Del Poeta, Maurizio</contributor><creatorcontrib>Moodley, Bhavani</creatorcontrib><creatorcontrib>Tempia, Stefano</creatorcontrib><creatorcontrib>Frean, John Andrew</creatorcontrib><title>Comparison of quantitative real-time PCR and direct immunofluorescence for the detection of Pneumocystis jirovecii</title><title>PloS one</title><addtitle>PLoS One</addtitle><description>Pneumocystis pneumonia (PCP) is a serious risk for HIV-positive patients. Asymptomatic infection or colonisation with P. jirovecii has been shown to occur frequently. PCR assays frequently identify such cases, due to their high sensitivity. Quantitative real-time PCR (qPCR) gene copy number cut-off values have been suggested to differentiate colonisation and infection; these need to be standardised for routine use. We compared the results of qPCR with an immunofluorescence assay (IFA) to determine a specific cut-off value.
From March 2005 through June 2009, induced sputum specimens were collected from adult patients who were clinically suspected of having PCP, at the Chris Hani Baragwanath Hospital in Gauteng, South Africa. Laboratory diagnosis of PCP was done by a conventional direct IFA and a qPCR assay. A receiver operating characteristic (ROC) analysis was performed to determine a suitable copy number cut-off value.
P. jirovecii was identified in 51% (156/305) and 67% (204/305) of specimens using IFA and qPCR, respectively. The cut-off value for the qPCR that best predicted the IFA results was 78 copies/5 μl (area under ROC curve 0.92). The sensitivity and specificity of qPCR using this cut-off was 94.6% and 89.1%, respectively, compared with the IFA.
The results of the ROC curve analysis indicate an excellent predictive value of the qPCR using the proposed cut-off. However, the IFA test is an imperfect gold standard and so this cut-off should not be used in isolation; clinical data should also contribute to the interpretation of the qPCR result.</description><subject>Adult</subject><subject>Analysis</subject><subject>Assaying</subject><subject>Asymptomatic infection</subject><subject>Biology and Life Sciences</subject><subject>Colonization</subject><subject>Copy number</subject><subject>Cysts</subject><subject>Deoxyribonucleic acid</subject><subject>Diagnosis</subject><subject>Disease control</subject><subject>DNA</subject><subject>Female</subject><subject>Fluorescent antibody technique</subject><subject>Fluorescent Antibody Technique, Direct - methods</subject><subject>Health aspects</subject><subject>Health risks</subject><subject>HIV</subject><subject>Human immunodeficiency virus</subject><subject>Humans</subject><subject>Immunofluorescence</subject><subject>Infections</subject><subject>Influenza</subject><subject>Laboratories</subject><subject>Male</subject><subject>Medicine and Health Sciences</subject><subject>Parasitic diseases</subject><subject>Patients</subject><subject>People and places</subject><subject>Pneumocystis carinii - genetics</subject><subject>Pneumocystis carinii - isolation & purification</subject><subject>Pneumonia</subject><subject>Pneumonia, Pneumocystis - diagnosis</subject><subject>Pneumonia, Pneumocystis - microbiology</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>Sensitivity</subject><subject>Sputum</subject><subject>Zoonoses</subject><issn>1932-6203</issn><issn>1932-6203</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2017</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><sourceid>BENPR</sourceid><sourceid>DOA</sourceid><recordid>eNqNk9tqGzEQhpfS0qRp36C0C4XSXtjVYVcr3RSC6cEQSEgPt0IrjWyZ3ZUjaU3z9pVjJ9glF0UXEtI3vzS_ZoriNUZTTBv8aeXHMKhuuvYDTBHmqObiSXGKBSUTRhB9erA-KV7EuEKoppyx58UJ4YxTJMhpEWa-X6vgoh9Kb8ubUQ3JJZXcBsoAqpsk10N5Nbsu1WBK4wLoVLq-Hwdvu9EHiBoGDaX1oUxLKA2kTLid2tUAY-_1bUwulisX_Aa0cy-LZ1Z1EV7t57Pi19cvP2ffJxeX3-az84uJZoKkCTUGt6Jl3BgkmOJAuNWN1VgoVVnbaA2YoVZbEC1ChrKGVCAM5wqBpkzQs-LtTnfd-Sj3dkWJBWasaXiFMzHfEcarlVwH16twK71y8m7Dh4VUITndgWwVr1FNsCKkqrCpBaEc81ZzAoqr1matz_vbxrYHk11JQXVHoscng1vKhd_IukaIVjQLfNgLBH8zQkyyd9ncrlMD-PHu3U1OsuIko-_-QR_Pbk8tVE7ADdbne_VWVJ7XFSWCsarO1PQRKg8DvdO5tqzL-0cBH48CMpPgT1qoMUY5_3H9_-zl72P2_QG7zLWXltF347aY4jFY7UAdfIwB7IPJGMlta9y7IbetIfetkcPeHH7QQ9B9L9C__L8LlQ</recordid><startdate>20170706</startdate><enddate>20170706</enddate><creator>Moodley, Bhavani</creator><creator>Tempia, Stefano</creator><creator>Frean, John Andrew</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>PRINS</scope><scope>PTHSS</scope><scope>PYCSY</scope><scope>RC3</scope><scope>7X8</scope><scope>5PM</scope><scope>DOA</scope><orcidid>https://orcid.org/0000-0002-2344-3811</orcidid></search><sort><creationdate>20170706</creationdate><title>Comparison of quantitative real-time PCR and direct immunofluorescence for the detection of Pneumocystis jirovecii</title><author>Moodley, Bhavani ; Tempia, Stefano ; Frean, John Andrew</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c692t-3dd1b9b68dd096a8e28fc7fc19aa4ff7cce160bcfe9b00d36724e9d88a0ec3693</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2017</creationdate><topic>Adult</topic><topic>Analysis</topic><topic>Assaying</topic><topic>Asymptomatic infection</topic><topic>Biology and Life Sciences</topic><topic>Colonization</topic><topic>Copy number</topic><topic>Cysts</topic><topic>Deoxyribonucleic acid</topic><topic>Diagnosis</topic><topic>Disease control</topic><topic>DNA</topic><topic>Female</topic><topic>Fluorescent antibody technique</topic><topic>Fluorescent Antibody Technique, Direct - 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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>Moodley, Bhavani</au><au>Tempia, Stefano</au><au>Frean, John Andrew</au><au>Del Poeta, Maurizio</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Comparison of quantitative real-time PCR and direct immunofluorescence for the detection of Pneumocystis jirovecii</atitle><jtitle>PloS one</jtitle><addtitle>PLoS One</addtitle><date>2017-07-06</date><risdate>2017</risdate><volume>12</volume><issue>7</issue><spage>e0180589</spage><epage>e0180589</epage><pages>e0180589-e0180589</pages><issn>1932-6203</issn><eissn>1932-6203</eissn><abstract>Pneumocystis pneumonia (PCP) is a serious risk for HIV-positive patients. Asymptomatic infection or colonisation with P. jirovecii has been shown to occur frequently. PCR assays frequently identify such cases, due to their high sensitivity. Quantitative real-time PCR (qPCR) gene copy number cut-off values have been suggested to differentiate colonisation and infection; these need to be standardised for routine use. We compared the results of qPCR with an immunofluorescence assay (IFA) to determine a specific cut-off value.
From March 2005 through June 2009, induced sputum specimens were collected from adult patients who were clinically suspected of having PCP, at the Chris Hani Baragwanath Hospital in Gauteng, South Africa. Laboratory diagnosis of PCP was done by a conventional direct IFA and a qPCR assay. A receiver operating characteristic (ROC) analysis was performed to determine a suitable copy number cut-off value.
P. jirovecii was identified in 51% (156/305) and 67% (204/305) of specimens using IFA and qPCR, respectively. The cut-off value for the qPCR that best predicted the IFA results was 78 copies/5 μl (area under ROC curve 0.92). The sensitivity and specificity of qPCR using this cut-off was 94.6% and 89.1%, respectively, compared with the IFA.
The results of the ROC curve analysis indicate an excellent predictive value of the qPCR using the proposed cut-off. However, the IFA test is an imperfect gold standard and so this cut-off should not be used in isolation; clinical data should also contribute to the interpretation of the qPCR result.</abstract><cop>United States</cop><pub>Public Library of Science</pub><pmid>28683092</pmid><doi>10.1371/journal.pone.0180589</doi><tpages>e0180589</tpages><orcidid>https://orcid.org/0000-0002-2344-3811</orcidid><oa>free_for_read</oa></addata></record> |
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| subjects | Adult Analysis Assaying Asymptomatic infection Biology and Life Sciences Colonization Copy number Cysts Deoxyribonucleic acid Diagnosis Disease control DNA Female Fluorescent antibody technique Fluorescent Antibody Technique, Direct - methods Health aspects Health risks HIV Human immunodeficiency virus Humans Immunofluorescence Infections Influenza Laboratories Male Medicine and Health Sciences Parasitic diseases Patients People and places Pneumocystis carinii - genetics Pneumocystis carinii - isolation & purification Pneumonia Pneumonia, Pneumocystis - diagnosis Pneumonia, Pneumocystis - microbiology Polymerase chain reaction Real time Real-Time Polymerase Chain Reaction - methods Research and Analysis Methods Sensitivity Sputum Zoonoses |
| title | Comparison of quantitative real-time PCR and direct immunofluorescence for the detection of Pneumocystis jirovecii |
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