Primary Quantitative Reference Standards for Viral Nucleic Acids Should Be Developed Using Digital Polymerase Chain Reaction Instead of Consensus Testing.

Abstract

Quantitative molecular methods are the cornerstone for managing a variety of infectious diseases, including, but not limited to, human immunodeficiency virus type 1, hepatitis C virus, cytomegalovirus and BK polyomavirus. Currently, the field relies on World Health Organization (WHO)-approved international standards (IS) for the calibration of different assay platforms and for the reporting of results in international units (IU)/mL. This approach has been shown to improve the agreement of viral load values across different tests. The procedure for developing these primary standards relies on a consensus process, in which the amount of analyte in the specimen is determined via testing in a large number of laboratories (30 to 40), using a variety of testing platforms. While this method is well-established, it is quite time-consuming, requiring several years for a WHO IS (whether as an initial offering or as a new lot) to be available to diagnostic companies and to companies producing secondary and working standards. These standards are not traceable to a recognized reference method. Furthermore, the criteria for the selection of participating consensus laboratories are variably described as geographic distribution, expertise or experience in the detection of a given pathogen, or participation in prior National Institute for Biological Standards and Control (NIBSC) evaluations. These criteria may or may not necessarily select for those running assays with the highest accuracy. The question is whether the time has come to streamline and further improve this process by using digital polymerase chain reaction (PCR) to determine the concentrations of primary standards and thereby provide absolute quantification and reduce the time required to develop primary standards.