Detection of human herpesvirus 8 by quantitative polymerase chain reaction: development and standardisation of methods
<p>Abstract</p> <p>Background</p> <p>Human herpesvirus 8 (HHV-8), the aetiological agent of Kaposi’s sarcoma (KS), multicentric Castleman’s disease (MCD), and primary effusion lymphoma (PEL) is rare in Australia, but endemic in Sub-Saharan Africa, parts of South-east As...
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BMC
2012-09-01
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| Series: | BMC Infectious Diseases |
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| Online Access: | http://www.biomedcentral.com/1471-2334/12/210 |
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| author | Speicher David J Johnson Newell W |
| author_facet | Speicher David J Johnson Newell W |
| author_sort | Speicher David J |
| collection | DOAJ |
| description | <p>Abstract</p> <p>Background</p> <p>Human herpesvirus 8 (HHV-8), the aetiological agent of Kaposi’s sarcoma (KS), multicentric Castleman’s disease (MCD), and primary effusion lymphoma (PEL) is rare in Australia, but endemic in Sub-Saharan Africa, parts of South-east Asia and Oceania. While the treatment of external KS lesions can be monitored by clinical observation, the internal lesions of KS, MCD and PEL require extensive and expensive internal imaging, or autopsy. In patients with MCD and PEL, if HHV-8 viraemia is not reduced quickly, ~50% die within 24 months. HHV-8 qPCR is a valuable tool for monitoring HHV-8 viraemia, but is not available in many parts of the world, including those with high prevalence of KS and HHV-8.</p> <p>Methods</p> <p>A new molecular facility with stringent three-phase workflow was established, adhering to NPAAC and CLSI guidelines. Three fully validated quantitative assays were developed: two for detection and quantification of HHV-8; one for GAPDH, necessary for normalisation of viral loads in tissue and peripheral blood.</p> <p>Results</p> <p>The HHV-8 ORF73 and ORF26 qPCR assays were 100% specific. All qPCR assays, displayed a broad dynamic range (10<sup>2</sup> to 10<sup>10</sup> copies/μL TE Buffer) with a limit of detection of 4.85x10<sup>3</sup>, 5.61x10<sup>2</sup>, and 2.59x10<sup>2</sup> copies/μL TE Buffer and a limit of quantification of 4.85x10<sup>3</sup>, 3.01x10<sup>2</sup>, and 1.38x10<sup>2</sup> copies/μL TE Buffer for HHV-8 ORF73, HHV-8 ORF26, and GAPDH respectively.</p> <p>The assays were tested on a panel of 35 KS biopsies from Queensland. All were HHV-8 qPCR positive with average viral load of 2.96x10<sup>5</sup> HHV-8 copies/μL DNA extract (range: 4.37x10<sup>3</sup> to 1.47x10<sup>6</sup> copies/μL DNA extract): When normalised these equate to an average viral load of 2.44x10<sup>4</sup> HHV-8 copies/10<sup>3</sup> cells (range: 2.20x10<sup>2</sup> to 7.38x10<sup>5</sup> HHV-8 copies/10<sup>3</sup> cells).</p> <p>Conclusions</p> <p>These are the first fully optimised, validated and MIQE compliant HHV-8 qPCR assays established in Australia. They worked well for qualitative detection of HHV-8 in archival tissue, and are well-suited for quantitative detection in whole blood. They are now available for research, for clinical diagnosis of HHV-8 infection, and for monitoring treatment efficacy.</p> |
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| spelling | doaj.art-bd5b62baf8114ede889b6a112a00e4d32022-12-22T00:16:51ZengBMCBMC Infectious Diseases1471-23342012-09-0112121010.1186/1471-2334-12-210Detection of human herpesvirus 8 by quantitative polymerase chain reaction: development and standardisation of methodsSpeicher David JJohnson Newell W<p>Abstract</p> <p>Background</p> <p>Human herpesvirus 8 (HHV-8), the aetiological agent of Kaposi’s sarcoma (KS), multicentric Castleman’s disease (MCD), and primary effusion lymphoma (PEL) is rare in Australia, but endemic in Sub-Saharan Africa, parts of South-east Asia and Oceania. While the treatment of external KS lesions can be monitored by clinical observation, the internal lesions of KS, MCD and PEL require extensive and expensive internal imaging, or autopsy. In patients with MCD and PEL, if HHV-8 viraemia is not reduced quickly, ~50% die within 24 months. HHV-8 qPCR is a valuable tool for monitoring HHV-8 viraemia, but is not available in many parts of the world, including those with high prevalence of KS and HHV-8.</p> <p>Methods</p> <p>A new molecular facility with stringent three-phase workflow was established, adhering to NPAAC and CLSI guidelines. Three fully validated quantitative assays were developed: two for detection and quantification of HHV-8; one for GAPDH, necessary for normalisation of viral loads in tissue and peripheral blood.</p> <p>Results</p> <p>The HHV-8 ORF73 and ORF26 qPCR assays were 100% specific. All qPCR assays, displayed a broad dynamic range (10<sup>2</sup> to 10<sup>10</sup> copies/μL TE Buffer) with a limit of detection of 4.85x10<sup>3</sup>, 5.61x10<sup>2</sup>, and 2.59x10<sup>2</sup> copies/μL TE Buffer and a limit of quantification of 4.85x10<sup>3</sup>, 3.01x10<sup>2</sup>, and 1.38x10<sup>2</sup> copies/μL TE Buffer for HHV-8 ORF73, HHV-8 ORF26, and GAPDH respectively.</p> <p>The assays were tested on a panel of 35 KS biopsies from Queensland. All were HHV-8 qPCR positive with average viral load of 2.96x10<sup>5</sup> HHV-8 copies/μL DNA extract (range: 4.37x10<sup>3</sup> to 1.47x10<sup>6</sup> copies/μL DNA extract): When normalised these equate to an average viral load of 2.44x10<sup>4</sup> HHV-8 copies/10<sup>3</sup> cells (range: 2.20x10<sup>2</sup> to 7.38x10<sup>5</sup> HHV-8 copies/10<sup>3</sup> cells).</p> <p>Conclusions</p> <p>These are the first fully optimised, validated and MIQE compliant HHV-8 qPCR assays established in Australia. They worked well for qualitative detection of HHV-8 in archival tissue, and are well-suited for quantitative detection in whole blood. They are now available for research, for clinical diagnosis of HHV-8 infection, and for monitoring treatment efficacy.</p>http://www.biomedcentral.com/1471-2334/12/210HHV-8Kaposi’s sarcomaHuman herpesvirus 8Molecular diagnosticsLaboratory establishment |
| spellingShingle | Speicher David J Johnson Newell W Detection of human herpesvirus 8 by quantitative polymerase chain reaction: development and standardisation of methods BMC Infectious Diseases HHV-8 Kaposi’s sarcoma Human herpesvirus 8 Molecular diagnostics Laboratory establishment |
| title | Detection of human herpesvirus 8 by quantitative polymerase chain reaction: development and standardisation of methods |
| title_full | Detection of human herpesvirus 8 by quantitative polymerase chain reaction: development and standardisation of methods |
| title_fullStr | Detection of human herpesvirus 8 by quantitative polymerase chain reaction: development and standardisation of methods |
| title_full_unstemmed | Detection of human herpesvirus 8 by quantitative polymerase chain reaction: development and standardisation of methods |
| title_short | Detection of human herpesvirus 8 by quantitative polymerase chain reaction: development and standardisation of methods |
| title_sort | detection of human herpesvirus 8 by quantitative polymerase chain reaction development and standardisation of methods |
| topic | HHV-8 Kaposi’s sarcoma Human herpesvirus 8 Molecular diagnostics Laboratory establishment |
| url | http://www.biomedcentral.com/1471-2334/12/210 |
| work_keys_str_mv | AT speicherdavidj detectionofhumanherpesvirus8byquantitativepolymerasechainreactiondevelopmentandstandardisationofmethods AT johnsonnewellw detectionofhumanherpesvirus8byquantitativepolymerasechainreactiondevelopmentandstandardisationofmethods |