Comparative evaluation of a laboratory developed real-time PCR assay and the RealStar^® HHV-6 PCR Kit for quantitative detection of human herpesvirus 6

Background HHV-6 reactivation in immunocompromised patients is common and may be associated with serious morbidity and mortality; therefore, early detection and initiation of therapy might be of benefit. Real-time PCR assays allow for early identification of HHV-6 reactivation to assist in providing a timely response. Thus, we compared the performance of an in-house developed HHV-6 quantitative PCR assay with a commercially available kit, the RealStar^® HHV-6 PCR Kit. Method The analytical sensitivity, analytical specificity, linearity, precision and accuracy of the in-house developed HHV-6 qPCR assay were evaluated. The diagnostic performance of the in-house HHV-6 qPCR assay was compared with the RealStar^® HHV-6 PCR Kit, using 72 clinical specimens and 17 proficiency testing samples. Results Linear regression analysis of the quantitative results showed a dynamic range from 2 to 10 log₁₀ copies/ml and a coefficient of determination (R²) of 0.999 for the in-house assay. A dilution series demonstrated a limit of detection and a limit of quantification of 1.7 log₁₀ and 2 log₁₀ copies/ml, respectively. The precision of the assay was highly reproducible among runs with coefficients of variance (CV) ranging from 0.27% to 4.37%. A comparison of 27 matched samples showed an excellent correlation between the quantitative viral loads measured by the in-house HHV-6 qPCR assay and the RealStar^® HHV-6 PCR Kit (R² = 0.926; P < 0.0001), with an average bias of −0.24 log₁₀ copies/ml. Conclusions The in-house developed HHV-6 qPCR method is a sensitive and reliable assay with lower cost for the detection and quantification of HHV-6 DNA when compared to the RealStar^® HHV-6 PCR Kit.