Calibrated Real-Time PCR for Evaluation of Parvovirus B19 Viral Load

Parvovirus B19, a pathogenic virus widely distributed in the human population, is responsible for various pathologies and diverse clinical manifestations (1). Diagnosis by detection of virus and quantitative evaluation of viral load is important mainly at the onset of infections before an immune response develops, in cases of atypical immune response, in the course of chronic infections, and in the occurrence of fetal infections (2). In the acute phase of infections, virus can be present in the blood at concentrations >1015 virions/L, posing a risk of transmission through transfusions and therapeutic use of blood products. Quantitative evaluation of B19 virus contamination of plasma pools for the production of blood derivatives is required as a measure to reduce the risk of transmission of infections (3).Fluorescence-based real-time PCR assays can combine specific detection and quantitative evaluation of the viral load. For quantitative evaluation of the viral load, real-time PCR assays must be carefully designed to give reliable results. In particular, the mode of quantification, whether absolute or relative, and an appropriate calibration method need to be firmly established (4)(5). Absolute quantification can be obtained by referring to a calibration curve. To take into account the variability attributable to cumulating effects both in sample processing and in the analytical phases, the calculated amount of target nucleic acids can be normalized to the calculated amount of a reference target that is likely to be present in constant amounts in all samples analyzed. Alternatively, relative quantification can be obtained from the ratio of target present in experimental samples to target in a calibrator sample. To compensate for sample variability, ratios can be further normalized with respect to a parallel, relative quantification of a reference target (6).Different real-time PCR assays for the detection of B19 virus in clinical samples …