Diagnostic Assay Development for Poliovirus Eradication

Abstract

With poliovirus eradication nearing, few pockets of active wild poliovirus (WPV) transmission remain in the world. Intratypic differentiation (ITD) plays a crucial part in laboratory surveillance as the molecular detection method that can identify and distinguish wild and vaccine-like polioviruses isolated from acute flaccid paralysis cases or environmental sources. The need to detect new variants of WPV serotype 1 (WPV1) and the containment of all serotype 2 polioviruses (PV2) in 2015 required changes to the previous version of the method. The ITD version 5.0 is a set of six real-time reverse transcription-PCR (rRT-PCR) assays that serve as accurate diagnostic tools to easily detect and differentiate PV serotypes and genotypes. We describe the creation and properties of quantitation standards, including 16 control RNA transcripts and nine plaque-isolated viruses. All ITD rRT-PCR assays were validated using these standards, and the limits of detection were determined for each assay. We designed and pilot tested two new assays targeting recently circulating WPV1 genotypes and all PV2 viruses. The WPV1 assay had 99.1% specificity and 100% sensitivity, and the PV2 assay had 97.7% specificity and 92% sensitivity. Before proceeding to the next step in the global poliovirus eradication program, we needed to gain a better understanding of the performance of the ITD 5.0 suite of molecular assays and their limits of detection and specificities. The findings and conclusions in this evaluation serve as building blocks for future development work.

Keywords: diagnostic assay; molecular typing; polio eradication; poliovirus; rRT-PCR; real-time reverse transcription-PCR; serotype identification.

FIG 1

Sensitivity of detection of plaque-purified polioviruses (CCID₅₀ per microliter) in the ITD 5.0 assays by target assay and template. The color code indicates sensitivity of ≥95% (blue) or <95% (red) (calculated as a percentage of positive samples per dilution; n = 20).

FIG 2

Sensitivity of detection of synthetic RNA transcripts (RNA copy number per microliter) in the ITD 5.0 assays by target assay and template. The color code indicates sensitivity of ≥95% (blue) or <95% (red) (calculated as a percentage of positive samples per dilution; n = 20).

FIG 3

PV2 assay performance by participating pilot testing laboratory and reference viruses tested. Each dot represents a single virus tested against the PV2 assay. Colors indicate the participating laboratory: Centers for Disease Control and Prevention, United States (red; n = 200), Enterovirus Research Centre, India (blue; n = 149), and National Institute for Communicable Diseases (NICD), South Africa (green; n = 40).