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.

Real-time reverse transcription-polymerase chain reaction assays for identification of wild poliovirus 1 & 3.

BACKGROUND & OBJECTIVES: The poliovirus serotype identification and intratypic differentiation by real-time reverse transcription-polymerase chain reaction (rRT-PCR) assay is suitable for serotype mixtures but not for intratypic mixtures of wild and vaccine poliovirus strains. This study was undertaken to develop wild poliovirus 1 and 3 (WPV1 and WPV3) specific rRT-PCR assays for use. METHODS: Specific primers and probes for rRT-PCR were designed based on VP1 sequences of WPV1 and WPV3 isolated in India since 2000. The specificity of the rRT-PCR assays was evaluated using WPV1 and WPV3 of different genetic lineages, non-polio enteroviruses (NPEVs) and mixtures of wild/wild and wild/Sabin vaccine strains. The sensitivity of the assays was determined by testing serial 10-fold dilutions of wild poliovirus 1 and 3 stock suspensions of known titre. RESULTS: No cross-reactivity with Sabin strains, intertypic wild poliovirus isolates or 27 types of NPEVs across all the four Enterovirus species was found for both the wild poliovirus 1 and 3 rRT-PCR assays. All WPV1 and WPV3 strains isolated since 2000 were successfully amplified. The rRT-PCR assays detected 10 4.40 CCID 50 /ml of WPV1 and 10 4.00 CCID 50 /ml of WPV3, respectively either as single isolate or mixture with Sabin vaccine strains or intertypic wild poliovirus. INTERPRETATION & CONCLUSIONS: rRT-PCR assays for WPV1 and WPV3 have been validated to detect all the genetic variations of the WPV1 and WPV3 isolated in India for the last decade. When used in combination with the current rRT-PCR assay testing was complete for confirmation of the presence of wild poliovirus in intratypic mixtures.

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.

Genomic characterization of two new enterovirus types, EV-A114 and EV-A121.

Enteroviruses cause a variety of illnesses of the gastrointestinal tract, central nervous system and cardiovascular system. Phylogenetic analysis of VP1 sequences has identified 106 different human enteroviruses classified into four enterovirus species within the genus Enterovirus of the family Picornaviridae. It is likely that not all enterovirus types have been discovered. Between September 2013 and October 2014, stool samples of 6274 apparently healthy children of up to 5 years of age residing in Gorakhpur district, Uttar Pradesh, India were screened for enteroviruses. Virus isolates obtained in RD and Hep-2c cells were identified by complete VP1 sequencing. Enteroviruses were isolated from 3042 samples. A total of 87 different enterovirus types were identified. Two isolates with 71 and 74 % nucleotide sequence similarity to all other known enteroviruses were recognized as novel types. In this paper we report identification and complete genome sequence analysis of these two isolates classified as EV-A114 and EV-A121.

Serum IgG and IgA levels in polio and non-polio acute flaccid paralysis cases in western Uttar Pradesh, India.

OBJECTIVE: IgG and IgA immunocompetence of children with wild poliovirus poliomyelitis and non-polio acute flaccid paralysis. METHODS: 932 cases of acute flaccid paralysis, reported in 2008-2009, were tested for presence of polio and non-polio enteroviruses according to the WHO standards. Serum IgA and IgG levels were determined by sandwich ELISA. RESULTS: Mean (SD) IgA levels [0.87 (0.62)g/L; n=28] of virologically confirmed poliomyelitis cases were lower than those of virus negative [1.21 (0.83)g/L; n=612] and non-polio Enterovirus positive [1.22 (0.79)g/L; n=240] cases of acute flaccid paralysis. No significant difference was observed in the concentration of IgG among these groups. CONCLUSIONS: IgA plays an important role in protection against poliomyelitis.