Time to revisit the endpoint dilution assay and to replace TCID$_{50}$ and PFU as measures of a virus sample's infection concentration

Abstract

The infectivity of a virus sample is measured by the infections it causes, via a plaque or focus forming assay (PFU or FFU) or an endpoint dilution (ED) assay (TCID50, CCID50, EID50, etc., hereafter collectively ID50). The counting of plaques or foci at a given dilution intuitively and directly provides the concentration of infectious doses in the undiluted sample. However, it has many technical and experimental limitations. For example, it is subjective as it relies on one's judgement in distinguishing between two merged plaques and a larger one, or between small plaques and staining artifacts. In this regard, ED assays are more robust because one need only determine whether or not infection occurred. The output of the ED assay, the 50% infectious dose (ID50), is calculated using either the Spearman-Kärber (1908|1931) or Reed-Muench (1938) mathematical approximations. However, these are often miscalculated and their approximation of the ID50 cannot be reliably related to the infectious dose. Herein, we propose that the plaque and focus forming assays be abandoned, and that the measured output of the ED assay, the ID50, be replaced by a more useful measure we coined specific infections (SIN). We introduce a free, open-source webapplication, midSIN, that computes the SIN concentration in a virus sample from a standard ED assay, requiring no changes to current experimental protocols. We use midSIN to analyze sets of influenza and respiratory syncytial virus samples, and demonstrate that the SIN/mL of a sample reliably corresponds to the number of infections a sample will cause per unit volume. The SIN/mL concentration of a virus sample estimated by midSIN, unlike the ID50/mL, can be used directly to achieve the desired multiplicity of infection. Estimates obtained with midSIN are shown to be more accurate and robust than those obtained using the Reed-Muench and Spearman-Kärber approximations. The impact of ED plate design choices (dilution factor, replicates per dilution) on measurement accuracy is also explored. The simplicity of SIN as a measure and the greater accuracy provided by midSIN make them an easy and superior replacement for the PFU, FFU, TCID50 and other ID50 measures. We hope to see their universal adoption to measure the infectivity of virus samples.