Utility of Quantitative Polymerase Chain Reaction in Leptospirosis Diagnosis: Association of Level of Leptospiremia and Clinical Manifestations in Sri Lanka
Background. Quantitative polymerase chain reaction (qPCR), despite cost and logistical challenges, has the potential to provide accurate and timely diagnosis for leptospirosis at the point-of-care in endemic areas. We studied optimal sample types for qPCR, timing of sampling, and clinical manifestat...
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| Veröffentlicht in: | Clinical infectious diseases 2012-05, Vol.54 (9), p.1249-1255 |
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| creator | Agampodi, Suneth B. Matthias, Michael A. Moreno, Angelo C. Vinetz, Joseph M. |
| description | Background. Quantitative polymerase chain reaction (qPCR), despite cost and logistical challenges, has the potential to provide accurate and timely diagnosis for leptospirosis at the point-of-care in endemic areas. We studied optimal sample types for qPCR, timing of sampling, and clinical manifestations in relation to quantitative leptospiremia. Methods. A new qPCR assay using pathogenic Leptospira-specific 16S ribosomal RNA (rRNA) gene Taqman primers and an optimized temperature stepdown protocol was used to analyze patient blood samples. Serum was compared with whole blood as sample source. Quantitative leptospiremia was compared with clinical manifestations of leptospirosis and outcome. Results. The diagnostic sensitivity of qPCR of whole blood and serum was 18.4% (95% confidence interval [CI]: 9.97%-31.4%) and 51.0% (95% CI: 37.5%-64.4%) respectively. The qPCR on suspected cases confirmed infection in 58 of 381 cases (15.2%). Of these, 6 cases confirmed by nested polymerase chain reaction (PCR) and sequencing were serologically negative using a standard but not regionally optimized microscopic agglutination test panel. The bacterial load in serum/blood ranged from 10 2 to 10 6 Leptospira/mL. Median leptospiral load for uncomplicated, renal failure, myocarditis, and multi-organ failure patients were 8616, 11 007, 36 100, and 15 882 Leptospira/mL respectively. The qPCR window of positivity ranged from day 2 to day 15; sensitivity of qPCR was not affected by the length of the interval between the onset of symptoms and sample collection (P = .328). Conclusions. Quantitative PCR shows potential as a valid diagnostic test with a wider window of positivity than previously thought. Quantitative leptospiremia in serum/whole blood samples did not directly correlate with clinical manifestations of outcome in this patient population. |
| doi_str_mv | 10.1093/cid/cis035 |
| format | Article |
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Quantitative polymerase chain reaction (qPCR), despite cost and logistical challenges, has the potential to provide accurate and timely diagnosis for leptospirosis at the point-of-care in endemic areas. We studied optimal sample types for qPCR, timing of sampling, and clinical manifestations in relation to quantitative leptospiremia. Methods. A new qPCR assay using pathogenic Leptospira-specific 16S ribosomal RNA (rRNA) gene Taqman primers and an optimized temperature stepdown protocol was used to analyze patient blood samples. Serum was compared with whole blood as sample source. Quantitative leptospiremia was compared with clinical manifestations of leptospirosis and outcome. Results. The diagnostic sensitivity of qPCR of whole blood and serum was 18.4% (95% confidence interval [CI]: 9.97%-31.4%) and 51.0% (95% CI: 37.5%-64.4%) respectively. The qPCR on suspected cases confirmed infection in 58 of 381 cases (15.2%). Of these, 6 cases confirmed by nested polymerase chain reaction (PCR) and sequencing were serologically negative using a standard but not regionally optimized microscopic agglutination test panel. The bacterial load in serum/blood ranged from 10 2 to 10 6 Leptospira/mL. Median leptospiral load for uncomplicated, renal failure, myocarditis, and multi-organ failure patients were 8616, 11 007, 36 100, and 15 882 Leptospira/mL respectively. The qPCR window of positivity ranged from day 2 to day 15; sensitivity of qPCR was not affected by the length of the interval between the onset of symptoms and sample collection (P = .328). Conclusions. Quantitative PCR shows potential as a valid diagnostic test with a wider window of positivity than previously thought. Quantitative leptospiremia in serum/whole blood samples did not directly correlate with clinical manifestations of outcome in this patient population.</description><identifier>ISSN: 1058-4838</identifier><identifier>EISSN: 1537-6591</identifier><identifier>DOI: 10.1093/cid/cis035</identifier><identifier>PMID: 22354922</identifier><identifier>CODEN: CIDIEL</identifier><language>eng</language><publisher>Oxford: Oxford University Press</publisher><subject>Agglutination Tests ; and Commentaries ; ARTICLES AND COMMENTARIES ; Bacteremia - diagnosis ; Bacteremia - epidemiology ; Bacteremia - microbiology ; Bacterial diseases ; Bacterial load ; Biological and medical sciences ; Blood ; Blood tests ; Clinical outcomes ; Cohort Studies ; Confidence Intervals ; Disease Outbreaks ; DNA Primers - genetics ; DNA, Bacterial - blood ; DNA, Bacterial - genetics ; Female ; Fever ; Health outcomes ; Human bacterial diseases ; Humans ; Infectious diseases ; Leptospira ; Leptospira - genetics ; Leptospira - isolation & purification ; Leptospirosis ; Leptospirosis - diagnosis ; Leptospirosis - epidemiology ; Leptospirosis - microbiology ; Male ; Medical diagnosis ; Medical sciences ; Miscellaneous ; Patients ; Polymerase chain reaction ; Quantification ; Real-Time Polymerase Chain Reaction - methods ; Real-Time Polymerase Chain Reaction - standards ; Retrospective Studies ; Ribonucleic acid ; RNA ; RNA, Ribosomal, 16S - genetics ; Sensitivity and Specificity ; Sequence Analysis, DNA ; Sequencing ; Specimens ; Sri Lanka - epidemiology</subject><ispartof>Clinical infectious diseases, 2012-05, Vol.54 (9), p.1249-1255</ispartof><rights>Copyright © 2012 Oxford University Press on behalf of the Infectious Diseases Society of America</rights><rights>2015 INIST-CNRS</rights><rights>Copyright University of Chicago, acting through its Press May 1, 2012</rights><rights>The Author 2012. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com. 2012</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c490t-ef268ad6b5d2443733f88d54a0da62b7f56f3ac7734961bb291d63475fb766e73</citedby><cites>FETCH-LOGICAL-c490t-ef268ad6b5d2443733f88d54a0da62b7f56f3ac7734961bb291d63475fb766e73</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.jstor.org/stable/pdf/23213323$$EPDF$$P50$$Gjstor$$H</linktopdf><linktohtml>$$Uhttps://www.jstor.org/stable/23213323$$EHTML$$P50$$Gjstor$$H</linktohtml><link.rule.ids>230,314,780,784,803,885,27924,27925,58017,58250</link.rule.ids><backlink>$$Uhttp://pascal-francis.inist.fr/vibad/index.php?action=getRecordDetail&idt=25816423$$DView record in Pascal Francis$$Hfree_for_read</backlink><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/22354922$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Agampodi, Suneth B.</creatorcontrib><creatorcontrib>Matthias, Michael A.</creatorcontrib><creatorcontrib>Moreno, Angelo C.</creatorcontrib><creatorcontrib>Vinetz, Joseph M.</creatorcontrib><title>Utility of Quantitative Polymerase Chain Reaction in Leptospirosis Diagnosis: Association of Level of Leptospiremia and Clinical Manifestations in Sri Lanka</title><title>Clinical infectious diseases</title><addtitle>Clin Infect Dis</addtitle><description>Background. Quantitative polymerase chain reaction (qPCR), despite cost and logistical challenges, has the potential to provide accurate and timely diagnosis for leptospirosis at the point-of-care in endemic areas. We studied optimal sample types for qPCR, timing of sampling, and clinical manifestations in relation to quantitative leptospiremia. Methods. A new qPCR assay using pathogenic Leptospira-specific 16S ribosomal RNA (rRNA) gene Taqman primers and an optimized temperature stepdown protocol was used to analyze patient blood samples. Serum was compared with whole blood as sample source. Quantitative leptospiremia was compared with clinical manifestations of leptospirosis and outcome. Results. The diagnostic sensitivity of qPCR of whole blood and serum was 18.4% (95% confidence interval [CI]: 9.97%-31.4%) and 51.0% (95% CI: 37.5%-64.4%) respectively. The qPCR on suspected cases confirmed infection in 58 of 381 cases (15.2%). Of these, 6 cases confirmed by nested polymerase chain reaction (PCR) and sequencing were serologically negative using a standard but not regionally optimized microscopic agglutination test panel. The bacterial load in serum/blood ranged from 10 2 to 10 6 Leptospira/mL. Median leptospiral load for uncomplicated, renal failure, myocarditis, and multi-organ failure patients were 8616, 11 007, 36 100, and 15 882 Leptospira/mL respectively. The qPCR window of positivity ranged from day 2 to day 15; sensitivity of qPCR was not affected by the length of the interval between the onset of symptoms and sample collection (P = .328). Conclusions. Quantitative PCR shows potential as a valid diagnostic test with a wider window of positivity than previously thought. Quantitative leptospiremia in serum/whole blood samples did not directly correlate with clinical manifestations of outcome in this patient population.</description><subject>Agglutination Tests</subject><subject>and Commentaries</subject><subject>ARTICLES AND COMMENTARIES</subject><subject>Bacteremia - diagnosis</subject><subject>Bacteremia - epidemiology</subject><subject>Bacteremia - microbiology</subject><subject>Bacterial diseases</subject><subject>Bacterial load</subject><subject>Biological and medical sciences</subject><subject>Blood</subject><subject>Blood tests</subject><subject>Clinical outcomes</subject><subject>Cohort Studies</subject><subject>Confidence Intervals</subject><subject>Disease Outbreaks</subject><subject>DNA Primers - genetics</subject><subject>DNA, Bacterial - blood</subject><subject>DNA, Bacterial - genetics</subject><subject>Female</subject><subject>Fever</subject><subject>Health outcomes</subject><subject>Human bacterial diseases</subject><subject>Humans</subject><subject>Infectious diseases</subject><subject>Leptospira</subject><subject>Leptospira - genetics</subject><subject>Leptospira - isolation & purification</subject><subject>Leptospirosis</subject><subject>Leptospirosis - diagnosis</subject><subject>Leptospirosis - epidemiology</subject><subject>Leptospirosis - microbiology</subject><subject>Male</subject><subject>Medical diagnosis</subject><subject>Medical sciences</subject><subject>Miscellaneous</subject><subject>Patients</subject><subject>Polymerase chain reaction</subject><subject>Quantification</subject><subject>Real-Time Polymerase Chain Reaction - methods</subject><subject>Real-Time Polymerase Chain Reaction - standards</subject><subject>Retrospective Studies</subject><subject>Ribonucleic acid</subject><subject>RNA</subject><subject>RNA, Ribosomal, 16S - genetics</subject><subject>Sensitivity and Specificity</subject><subject>Sequence Analysis, DNA</subject><subject>Sequencing</subject><subject>Specimens</subject><subject>Sri Lanka - epidemiology</subject><issn>1058-4838</issn><issn>1537-6591</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2012</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNp9kkuLFDEUhQtRnIdu3CsBGRShNe-kXAhD-4QSn7MOt6pSM-mpTtok3dD_xR9reqqdURcuQg7c7x7uTU5VPSD4OcE1e9G5vpyEmbhVHRLB1EyKmtwuGgs945rpg-oopQXGhGgs7lYHlDLBa0oPq59n2Y0ub1EY0Jc1-OwyZLex6HMYt0sbIVk0vwDn0VcLXXbBo6Ibu8ohrVwMySX02sG536mX6DSl0Dm44opjYzd2nMS-wS4dIPA9mo_Ouw5G9BG8G2zKV01p5_4tOtSAv4R71Z0BxmTv7-_j6uztm-_z97Pm07sP89Nm1vEa55kdqNTQy1b0lHOmGBu07gUH3IOkrRqEHBh0SjFeS9K2tCa9ZFyJoVVSWsWOq1eT72rdLm3fWZ8jjGYV3RLi1gRw5u-KdxfmPGwM45hLXReDJ3uDGH6syzJm6VJnxxG8Detk6prVWGlGCvn0vyQRRCpBmdpN9fgfdBHW0ZeHMARTqbgUWBfq2UR15TNStMP12ASbXTxMiYeZ4lHgR38ueo3-zkMBTvYApPI5QwRfWm84oYnklBXu4cQtUg7xps4oYazUfwHyS8_l</recordid><startdate>20120501</startdate><enddate>20120501</enddate><creator>Agampodi, Suneth B.</creator><creator>Matthias, Michael A.</creator><creator>Moreno, Angelo C.</creator><creator>Vinetz, Joseph M.</creator><general>Oxford University Press</general><scope>IQODW</scope><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>7QL</scope><scope>7T2</scope><scope>7T7</scope><scope>7U7</scope><scope>7U9</scope><scope>8FD</scope><scope>C1K</scope><scope>FR3</scope><scope>H94</scope><scope>K9.</scope><scope>M7N</scope><scope>P64</scope><scope>F1W</scope><scope>H95</scope><scope>H97</scope><scope>L.G</scope><scope>7X8</scope><scope>5PM</scope></search><sort><creationdate>20120501</creationdate><title>Utility of Quantitative Polymerase Chain Reaction in Leptospirosis Diagnosis: Association of Level of Leptospiremia and Clinical Manifestations in Sri Lanka</title><author>Agampodi, Suneth B. ; 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Quantitative polymerase chain reaction (qPCR), despite cost and logistical challenges, has the potential to provide accurate and timely diagnosis for leptospirosis at the point-of-care in endemic areas. We studied optimal sample types for qPCR, timing of sampling, and clinical manifestations in relation to quantitative leptospiremia. Methods. A new qPCR assay using pathogenic Leptospira-specific 16S ribosomal RNA (rRNA) gene Taqman primers and an optimized temperature stepdown protocol was used to analyze patient blood samples. Serum was compared with whole blood as sample source. Quantitative leptospiremia was compared with clinical manifestations of leptospirosis and outcome. Results. The diagnostic sensitivity of qPCR of whole blood and serum was 18.4% (95% confidence interval [CI]: 9.97%-31.4%) and 51.0% (95% CI: 37.5%-64.4%) respectively. The qPCR on suspected cases confirmed infection in 58 of 381 cases (15.2%). Of these, 6 cases confirmed by nested polymerase chain reaction (PCR) and sequencing were serologically negative using a standard but not regionally optimized microscopic agglutination test panel. The bacterial load in serum/blood ranged from 10 2 to 10 6 Leptospira/mL. Median leptospiral load for uncomplicated, renal failure, myocarditis, and multi-organ failure patients were 8616, 11 007, 36 100, and 15 882 Leptospira/mL respectively. The qPCR window of positivity ranged from day 2 to day 15; sensitivity of qPCR was not affected by the length of the interval between the onset of symptoms and sample collection (P = .328). Conclusions. Quantitative PCR shows potential as a valid diagnostic test with a wider window of positivity than previously thought. Quantitative leptospiremia in serum/whole blood samples did not directly correlate with clinical manifestations of outcome in this patient population.</abstract><cop>Oxford</cop><pub>Oxford University Press</pub><pmid>22354922</pmid><doi>10.1093/cid/cis035</doi><tpages>7</tpages><oa>free_for_read</oa></addata></record> |
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| subjects | Agglutination Tests and Commentaries ARTICLES AND COMMENTARIES Bacteremia - diagnosis Bacteremia - epidemiology Bacteremia - microbiology Bacterial diseases Bacterial load Biological and medical sciences Blood Blood tests Clinical outcomes Cohort Studies Confidence Intervals Disease Outbreaks DNA Primers - genetics DNA, Bacterial - blood DNA, Bacterial - genetics Female Fever Health outcomes Human bacterial diseases Humans Infectious diseases Leptospira Leptospira - genetics Leptospira - isolation & purification Leptospirosis Leptospirosis - diagnosis Leptospirosis - epidemiology Leptospirosis - microbiology Male Medical diagnosis Medical sciences Miscellaneous Patients Polymerase chain reaction Quantification Real-Time Polymerase Chain Reaction - methods Real-Time Polymerase Chain Reaction - standards Retrospective Studies Ribonucleic acid RNA RNA, Ribosomal, 16S - genetics Sensitivity and Specificity Sequence Analysis, DNA Sequencing Specimens Sri Lanka - epidemiology |
| title | Utility of Quantitative Polymerase Chain Reaction in Leptospirosis Diagnosis: Association of Level of Leptospiremia and Clinical Manifestations in Sri Lanka |
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