This article summarizes the status of environmental surveillance (ES) used by the Global Polio Eradication Initiative, provides the rationale for ES, gives examples of ES methods and findings, and summarizes how these data are used to achieve poliovirus eradication. ES complements clinical acute flaccid paralysis (AFP) surveillance for possible polio cases. ES detects poliovirus circulation in environmental sewage and is used to monitor transmission in communities. If detected, the genetic sequences of polioviruses isolated from ES are compared with those of isolates from clinical cases to evaluate the relationships among viruses. To evaluate poliovirus transmission, ES programs must be developed in a manner that is sensitive, with sufficiently frequent sampling, appropriate isolation methods, and specifically targeted sampling sites in locations at highest risk for poliovirus transmission. After poliovirus ceased to be detected in human cases, ES documented the absence of endemic WPV transmission and detected imported WPV. ES provides valuable information, particularly in high-density populations where AFP surveillance is of poor quality, persistent virus circulation is suspected, or frequent virus reintroduction is perceived. Given the benefits of ES, GPEI plans to continue and expand ES as part of its strategic plan and as a supplement to AFP surveillance.
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 (<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 >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.
BackgroundEnteroviruses are common human pathogens occasionally associated with severe disease, notoriously paralytic poliomyelitis caused by poliovirus. Other enterovirus serotypes such as enterovirus A71 and D68 have been linked to severe neurological syndromes. New enterovirus serotypes continue to emerge, some believed to be derived from nonhuman primates. However, little is known about the circulation patterns of many enterovirus serotypes and, in particular, the detailed enterovirus composition of sewage samples.MethodsWe used a next-generation sequencing approach analyzing reverse transcriptase polymerase chain reaction products synthesized directly from sewage concentrates.ResultsWe determined whole-capsid genome sequences of multiple enterovirus strains from all 4 A to D species present in environmental samples from the United Kingdom, Senegal, and Pakistan.ConclusionsOur results indicate complex enterovirus circulation patterns in human populations with differences in serotype composition between samples and evidence of sustained and widespread circulation of many enterovirus serotypes. Our analyses revealed known and divergent enterovirus strains, some of public health relevance and genetically linked to clinical isolates. Enteroviruses identified in sewage included vaccine-derived poliovirus and enterovirus D-68 stains, new enterovirus A71 and coxsackievirus A16 genogroups indigenous to Pakistan, and many strains from rarely reported serotypes. We show how this approach can be used for the early detection of emerging pathogens and to improve our understanding of enterovirus circulation in humans.
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