Rapid and Sensitive Direct Detection and Identification of Poliovirus from Stool and Environmental Surveillance Samples by Use of Nanopore Sequencing

Abstract

<jats:p>Global poliovirus surveillance involves virus isolation from stool and environmental samples, intratypic differential (ITD) by PCR and sequencing of the VP1 region to distinguish vaccine (Sabin), vaccine-derived and wild-type polioviruses and ensure an appropriate response. This cell-culture algorithm takes 2-3 weeks on average between sample receipt and sequencing. Direct detection of viral RNA using PCR allows faster detection but has traditionally faced challenges related to poor sensitivity and difficulties in sequencing common samples containing poliovirus and enterovirus mixtures. We present a nested PCR and nanopore sequencing protocol that allows rapid (&lt;3 days) and sensitive direct detection and sequencing of polioviruses in stool and environmental samples. We developed barcoded primers and a real-time analysis platform that generate accurate VP1 consensus sequences from multiplexed samples. The sensitivity and specificity compared with cell-culture were 90.9% (95% Confidence Interval: 75.7-98.1%) and 99.2% (95.5-100.0%) for wild-type 1 poliovirus, 92.5% (79.6-98.4%) and 98.7% (95.4-99.8%) for vaccine and vaccine-derived serotype 2 poliovirus, and 88.3% (81.2-93.5%) and 93.2% (88.6-96.3%) for Sabin 1 and 3 poliovirus alone or in mixtures when tested on 155 stool samples in Pakistan. Variant analysis of sequencing reads also allowed identification of polioviruses and enteroviruses in artificial mixtures and was able to distinguish complex mixtures of polioviruses in environmental samples. The median identity of consensus nanopore sequences with Sanger or Illumina sequences from the same samples was &gt;99.9%. This novel method shows promise as a faster and safer alternative to cell-culture for the detection and real-time sequencing of polioviruses in stool and environmental samples.</jats:p>

Diagnostic Efficiency of Three Fully Automated Serology Assays and Their Correlation with a Novel Surrogate Virus Neutralization Test in Symptomatic and Asymptomatic SARS-COV-2 Individuals

“…To assess the specificity of the automated assays, we have compiled pre-COVID-19 pandemic plasma sample obtained before the first appearance of the SARS-CoV-2 virus. Among the three assays, LIAISON ® XL showed the highest specificity (100%), similar to a previous study from the United States that reported a 99.9% specificity [ 48 ]. However, the sensitivity of LIAISON ® XL in our study using RT-PCR as the reference test was much lower (85.6%) compared to the one reported by the aforementioned study (100% by day 17 post symptoms onset) [ 48 ].…”

High Diversity of Human Non-Polio Enterovirus Serotypes Identified in Contaminated Water in Nigeria

“…Our study further illustrates the power of using next generation sequencing analysis allowing the identification of multiple EV strains in a single sample [ 14 , 15 , 16 , 17 , 18 , 19 ] and overcoming the limitations of traditional surveillance methods based on cell culture virus isolation and Sanger sequencing [ 49 ]. The number of serotypes identified in this study far exceeds those found in previous studies.…”

“…Although any amplification-based method such as the whole-capsid RT-PCR assay described here may contribute to bias toward specific strains or serotypes and may risk missing some strains due to mismatches in primer binding sequence regions, we have so far been able to sequence in different studies EV strains from 102 of the 110 different EV serotypes that have been described [ 16 , 17 , 19 ]. The use of environmental surveillance also contributes to expanding the diversity of EV serotypes that can be detected, improving our understanding of EVs circulating in human populations, as most EV infections appear to be asymptomatic or their detection requires timely and adequate sample collection and analysis.…”

“…Whole-capsid EV sequences present in water concentrates were determined by NGS analysis of whole-capsid PCR products using methods described in Section 2.2 , Section 2.3 and Section 2.4 . These methods have been extensively validated using reference EV strains, laboratory-made EV mixtures, clinical samples, and water concentrates from different locations in comparison to the sequences determined by the Sanger method [ 16 , 17 , 19 ].…”

“…There is plenty of evidence of ES being able to track down the interruption of poliovirus transmission detecting both vaccine viruses following immunization campaigns or vaccine cessation and wild-type or vaccine-derived polioviruses during outbreaks [ 13 ]. The use of molecular methods in combination with next generation sequencing (NGS) analysis directly from clinical and environmental samples, without the use of cell culture virus isolation, by us and others is starting to unravel the complexity of the human virome in wastewater samples [ 14 , 15 , 16 , 17 , 18 , 19 ].…”