Virtual quantification of influenza A virus load by real-time RT-PCR

Background The pan-influenza A real-time RT-PCR detection assay developed by the Centers for Disease Control and Prevention (CDC) during the 2009 pandemic is widely utilized. A quantitative version of the assay may be useful to monitor influenza A infection and response to treatment. Objectives To prove in principle the possibility that a virtual quantification tool (VQT) would allow conversion of CDC real-time RT-PCR cycle threshold (Ct) values in virus RNA copy number. Study design A plasmid carrying the CDC real-time RT-PCR target region of the influenza A Matrix (M) gene was generated. In a multicenter study, a set of 5 ten-fold dilutions (equivalent to 1 × 10 2 to 1 × 10 6 copies/reaction) were prepared and distributed to the 4 participating virology laboratories and then amplified to generate a virtual quantification standard curve. Clinical samples ( n = 120) were quantified in parallel by interpolation with locally generated standard curves and using the VQT. Results A total of 40 standard curves were obtained by the participating centers (ten from each center). The intra- and inter-laboratory variability showed a coefficient of variation (CV) ≤5%. Influenza A virus quantification in 120 respiratory samples showed a significant correlation between interpolation with locally generated standard curves and the VQT ( R 2 = 0.9655). Bland Altman analysis showed that the majority (no. 111, 92.5%) of clinical samples had 10 variation. Conclusions VQT proofs the concept that qualitative results from real-time RT-PCR assays can be converted into quantitative determination of virus load in clinical samples without running standard curves in parallel.