Abstract
BackgroundHuman 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.MethodsA 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.ResultsThe HHV-8 ORF73 and ORF26 qPCR assays were 100% specific. All qPCR assays, displayed a broad dynamic range (102 to 1010 copies/μL TE Buffer) with a limit of detection of 4.85x103, 5.61x102, and 2.59x102 copies/μL TE Buffer and a limit of quantification of 4.85x103, 3.01x102, and 1.38x102 copies/μL TE Buffer for HHV-8 ORF73, HHV-8 ORF26, and GAPDH respectively.The assays were tested on a panel of 35 KS biopsies from Queensland. All were HHV-8 qPCR positive with average viral load of 2.96x105 HHV-8 copies/μL DNA extract (range: 4.37x103 to 1.47x106 copies/μL DNA extract): When normalised these equate to an average viral load of 2.44x104 HHV-8 copies/103 cells (range: 2.20x102 to 7.38x105 HHV-8 copies/103 cells).ConclusionsThese 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.
Highlights
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
None of these laboratories perform quantitative PCR to monitor viral loads and treatment efficacy: This is of concern because, while the treatment of superficial KS can be monitored by clinical observation, both MCD and PEL require more extensive and expensive internal imaging, notably with computed axial tomography (CAT), magnetic resonance imaging (MRI) or positron emission tomography (PET) scans
Cloning of HHV-8 and glyceraldehyde 3phosphate dehydrogenase (GAPDH) constructs for quantitative PCR (qPCR) standards Amplicon containing –TA overhangs were produced by conventional PCR performed in a 20 μL volume containing 10X ThermoPol Reaction Buffer (New England BioLabsW Inc., Australia), 0.4 mM deoxyribonucleotide triphosphate, 0.2 μM primers (GeneWorks, Australia), 0.24 U/μL Taq DNA polymerase (New England BioLabsW Inc., Australia) and 2 μL of nucleic acid extract (DNeasy Blood and Tissue Kit, Qiagen, Australia)
Summary
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. HHV-8 nested PCR is performed at PathWest Laboratory Medicine, QEII Hospital, in Perth, Western Australia (the referral laboratory for Queensland Health), while real-time PCR (rtPCR) is performed at the Victorian Infectious Diseases Reference Laboratory (VIDRL) in Melbourne, Victoria and the South Eastern Area Laboratory Services (SEALS), Prince of Wales Hospital in Sydney, New South Wales None of these laboratories perform quantitative PCR (qPCR) to monitor viral loads and treatment efficacy: This is of concern because, while the treatment of superficial KS can be monitored by clinical observation, both MCD and PEL require more extensive and expensive internal imaging, notably with computed axial tomography (CAT), magnetic resonance imaging (MRI) or positron emission tomography (PET) scans
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
