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Validated Quantification of HHV-8 DNA Using Inter-Convertible Plasmid and Cell-Derived Calibrators: Optimization of a
Celeste Luján Pérez1, Carlos Ochoa Gamboa1, Mónica Tous1
1Tissue Culture Service, Virology Department, INEI-ANLIS "Dr. Carlos G. Malbrán" (1282AAF), Buenos Aires C1282AAF, Argentina.
Viruses
|May 27, 2026
Summary
A new quantitative PCR assay accurately measures Human Herpesvirus 8 (HHV-8) DNA in whole blood. This validated tool aids in diagnosing Kaposi
Area of Science:
- Virology
- Molecular Diagnostics
- Infectious Diseases
Background:
- Human herpesvirus 8 (HHV-8) is linked to Kaposi's sarcoma (KS), primary effusion lymphoma (PEL), and multicentric Castleman disease (MCD).
- Accurate quantification of HHV-8 DNA in whole blood is crucial for clinical management but lacks standardized laboratory methods.
- Existing laboratory practices for HHV-8 DNA quantification are inconsistent, hindering reliable patient assessment.
Purpose of the Study:
- To develop and validate a robust in-house quantitative PCR (qPCR) assay for precise HHV-8 DNA quantification in whole blood.
- To establish a reliable diagnostic tool for HHV-8 detection and monitoring across various clinical conditions.
- To address the heterogeneity in current laboratory practices for HHV-8 DNA measurement.
Main Methods:
- Developed a novel qPCR assay targeting the ORF26 gene, optimized specifically for whole blood samples.
- Calibrated the assay using diverse standards: plasmid DNA, BCBL-1 cell-derived DNA, and commercial HHV-8 DNA standards.
- Performed rigorous analytical and clinical validation according to CLSI and MIQE guidelines, assessing sensitivity, specificity, and reproducibility.
Main Results:
- The assay demonstrated high analytical performance with a 95% limit of detection of 65.7 copies/reaction and excellent efficiency (90-101%).
- Clinical validation achieved 100% sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) against a composite reference.
- Distinct viral load patterns were observed across different HHV-8-associated conditions, correlating with disease severity and response to treatment.
Conclusions:
- The validated qPCR assay offers a reliable, cost-effective method for quantifying HHV-8 DNA in whole blood.
- This assay can significantly improve diagnostic accuracy and patient monitoring for HHV-8-related diseases.
- The developed assay is suitable for integration into routine clinical workflows, supporting better patient care and research.
