Diagnostic Assay Development for Poliovirus Eradication.

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.

Culture-Independent Detection of Poliovirus in Stool Samples by Direct RNA Extraction.

Laboratory surveillance for poliovirus (PV) relies on virus isolation by cell culture to identify PV in stool specimens from acute flaccid paralysis (AFP) cases. Although this method successfully identifies PV, it is time-consuming and necessitates the additional biorisk of growing live virus in an increasingly polio-free world. To reduce the risk of culturing PV, the Global Polio Laboratory Network (GPLN) must switch to culture-independent diagnostic methods with sensitivity at least equivalent to that of cell culture procedures. Five commercial nucleic acid extraction kits and one enrichment method were tested for PV extraction efficiency. RNA yield was measured using real-time reverse transcription (RT)-PCR. Based on greater RNA yield, compared with the other kits, the Quick-RNA viral kit was selected for further testing and was optimized using an RNA extraction procedure for stool suspensions. RNA extraction was retrospectively tested with 182 stool samples that had previously tested positive for PVs, in parallel with the standard GPLN virus isolation algorithm. After virus isolation or RNA extraction, real-time RT-PCR assays were performed. RNA extraction was significantly more sensitive than virus isolation (McNemar's test, P < 0.001). Thereafter, the RNA extraction method was tested in parallel for 202 prospective samples; RNA extraction and virus isolation were not significantly different from each other (McNemar's test, P = 0.13). Direct RNA extraction was noninferior to current cell culture methods for detecting PV in stool samples. Our results show that direct RNA extraction can make downstream manipulation safer and can reduce the risk of accidental posteradication viral release. The method is amenable to implementation in a wide variety of polio laboratories. IMPORTANCE Successfully identifying poliovirus from acute flaccid paralysis (AFP) cases is a vital role of the Global Polio Laboratory Network to achieve the goals of the Global Polio Eradication Initiative. Currently, laboratory surveillance relies on virus isolation by cell culture to test for PV present in stool samples. Although this method can identify polioviruses, laboratories must switch to culture-independent methods to reduce the risk associated with growing live viruses in a soon-to-be polio-free world. By implementing this streamlined method, in combination with real-time RT-PCR, laboratories can quickly screen for and type polioviruses of programmatic importance to support the final stages of global polio eradication.

Direct detection of polioviruses using a recombinant poliovirus receptor.

Polioviruses are positive-sense, single-stranded RNA picornaviruses and the principal cause of poliomyelitis. Global poliovirus surveillance has relied on poliovirus isolation in cells, which may take a minimum of 10 days, involves maintaining two cell lines, and propagates virus in high titers. With eradication underway, a major objective of the Global Polio Eradication Initiative (GPEI) is to develop culture-independent detection of polioviruses as an alternative method to complement the current virus isolation technique. A culture-independent method on poliovirus-positive stool suspensions was assessed with commercially available recombinant soluble poliovirus receptor (PVR) coupled to Histidine (His) tags. Viral RNA was screened by quantitative real-time reverse transcription PCR using the poliovirus intratypic differentiation kit. Poliovirus recovery was optimized with PVR-His-tagged protein and buffers supplemented with polyethylene glycol. To validate the poliovirus-PVR-His tag purification assay, 182 poliovirus-positive stools of programmatic importance were parallel tested against the GPLN-accepted virus isolation method. The PVR-His tag enrichment method detected poliovirus in 164 of 171 poliovirus-positive stools, whereas the virus isolation method misidentified 38 stools as poliovirus-negative (McNemar χ2 p<0.0001). Using this method in combination with RNA extraction, viral RNA recovery increased and showed similar (WPV1) or higher (Sabin 1) sensitivity than the World Health Organization accredited variation of the virus isolation method. The PVR-His enrichment method could be a viable addition to poliovirus surveillance; similar methods have the potential to capture other human pathogens such as EV71 using an appropriate soluble His tag receptor.

Validation of a redesigned pan-poliovirus assay and real-time PCR platforms for the global poliovirus laboratory network.

Surveillance and detection of polioviruses (PV) remain crucial to monitoring eradication progress. Intratypic differentiation (ITD) using the real-time RT-PCR kit is key to the surveillance workflow, where viruses are screened after cell culture isolation before a subset are verified by sequencing. The ITD kit is a series of real-time RT-PCR assays that screens cytopathic effect (CPE)-positive cell cultures using the standard WHO method for virus isolation. Because ITD screening is a critical procedure in the poliovirus identification workflow, validation of performance of real-time PCR platforms is a core requirement for the detection of poliovirus using the ITD kit. In addition, the continual update and improvement of the ITD assays to simplify interpretation in all platforms is necessary to ensure that all real-time machines are capable of detecting positive real-time signals. Four platforms (ABI7500 real-time systems, Bio-Rad CFX96, Stratagene MX3000P, and the Qiagen Rotor-Gene Q) were validated with the ITD kit and a redesigned poliovirus probe. The poliovirus probe in the real-time RT-PCR pan-poliovirus (PanPV) assay was re-designed with a double-quencher (Zen™) to reduce background fluorescence and potential false negatives. The updated PanPV probe was evaluated with a panel consisting of 184 polioviruses and non-polio enteroviruses. To further validate the updated PanPV probe, the new assay was pilot tested in five Global Polio Laboratory Network (GPLN) laboratories (Madagascar, India, Philippines, Pakistan, and Democratic Republic of Congo). The updated PanPV probe performance was shown to reduce background fluorescence and decrease the number of false positives compared to the standard PanPV probe.

Rapid group-, serotype-, and vaccine strain-specific identification of poliovirus isolates by real-time reverse transcription-PCR using degenerate primers and probes containing deoxyinosine residues.

We have adapted our previously described poliovirus diagnostic reverse transcription-PCR (RT-PCR) assays to a real-time RT-PCR (rRT-PCR) format. Our highly specific assays and rRT-PCR reagents are designed for use in the WHO Global Polio Laboratory Network for rapid and large-scale identification of poliovirus field isolates.

Identification of vaccine-derived polioviruses using dual-stage real-time RT-PCR.

Vaccine-derived polioviruses (VDPVs) are associated with polio outbreaks and prolonged infections in individuals with primary immunodeficiencies. VDPV-specific PCR assays for each of the three Sabin oral poliovirus vaccine (OPV) strains were developed, targeting sequences within the VP1 capsid region that are selected for during replication of OPV in the human intestine. Over 2400 Sabin-related isolates and identified 755 VDPVs were screened. Sensitivity of all assays was 100%, while specificity was 100% for serotypes 1 and 3, and 76% for serotype 2. The assays permit rapid, sensitive identification of OPV-related viruses and flag programmatically important isolates for further characterization by genomic sequencing.

Poliovirus serotype-specific VP1 sequencing primers.

The Global Polio Laboratory Network routinely uses poliovirus-specific PCR primers and probes to determine the serotype and genotype of poliovirus isolates obtained as part of global poliovirus surveillance. To provide detailed molecular epidemiologic information, poliovirus isolates are further characterized by sequencing the ~900-nucleotide region encoding the major capsid protein, VP1. It is difficult to obtain quality sequence information when clinical or environmental samples contain poliovirus mixtures. As an alternative to conventional methods for resolving poliovirus mixtures, sets of serotype-specific primers were developed for amplifying and sequencing the VP1 regions of individual components of mixed populations of vaccine-vaccine, vaccine-wild, and wild-wild polioviruses.