Clinical correlation of influenza and respiratory syncytial virus load measured by digital PCR

Hijano, Diego R.; Brazelton de Cardenas, Jessica; Maron, Gabriela; Garner, Cherilyn D.; Ferrolino, Jose A.; Dallas, Ronald H.; Gu, Zhengming; Hayden, Randall T.

Viral load is commonly determined by quantification of viral genomic fragments [11] . Quantitative determinations by real-time PCR are indirect and provide a relative quantification of viral load [12] . However, the precision of such assays is limited by the nature of cycle threshold (Ct) determination with resulting values having a relative standard deviation that can exceed 50% [13] [14] [15] . In addition, assay workflow and design is complicated by the need for quantitative calibrators, introducing error risk and requirements for consistent supplies of well-characterized, commutable reference material. The latter can be particularly difficult to obtain for less commonly quantified viruses in general, and particularly for RNA viruses, such as those studied here [16] [17] [18] [19] [20] [21] . Marked variation in assay performance characteristics and in materials used as calibration has been described. The implementation of international standards, such as those that have been made available by the World Health Organization (WHO) has led to reduction of this problem. However, standards are only available for a limited number of pathogens and can vary over time [22, 23] .