Automated detection and classification of polioviruses from nanopore sequencing reads using piranha.

Widespread surveillance, rapid detection, and appropriate intervention will be critical for successful eradication of poliovirus. Using deployable next-generation sequencing (NGS) approaches, such as Oxford Nanopore Technologies' MinION, the time from sample to result can be significantly reduced compared to cell culture and Sanger sequencing. We developed piranha (poliovirus investigation resource automating nanopore haplotype analysis), a 'sequencing reads-to-report' solution to aid routine poliovirus testing of both stool and environmental samples and alleviate the bioinformatic bottleneck that often exists for laboratories adopting novel NGS approaches. Piranha can be used for efficient intratypic differentiation of poliovirus serotypes, for classification of Sabin-like polioviruses, and for detection of wild-type and vaccine-derived polioviruses. It produces interactive, distributable reports, as well as summary comma-separated values files and consensus poliovirus FASTA sequences. Piranha optionally provides phylogenetic analysis, with the ability to incorporate a local database, processing from raw sequencing reads to an interactive, annotated phylogeny in a single step. The reports describe each nanopore sequencing run with interpretable plots, enabling researchers to easily detect the presence of poliovirus in samples and quickly disseminate their results. Poliovirus eradication efforts are hindered by the lack of real-time detection and reporting, and piranha can be used to complement direct detection sequencing approaches.

Environmental Surveillance for Salmonella Typhi and its Association With Typhoid Fever Incidence in India and Malawi.

BACKGROUND: Environmental surveillance (ES) for Salmonella Typhi potentially offers a low-cost tool to identify communities with a high burden of typhoid fever. METHODS: We developed standardized protocols for typhoid ES, including sampling site selection, validation, characterization; grab or trap sample collection, concentration; and quantitative PCR targeting Salmonella genes (ttr, staG, and tviB) and a marker of human fecal contamination (HF183). ES was implemented over 12 months in a historically high typhoid fever incidence setting (Vellore, India) and a lower incidence setting (Blantyre, Malawi) during 2021-2022. RESULTS: S. Typhi prevalence in ES samples was higher in Vellore compared with Blantyre; 39/520 (7.5%; 95% confidence interval [CI], 4.4%-12.4%) vs 11/533 (2.1%; 95% CI, 1.1%-4.0%) in grab and 79/517 (15.3%; 95% CI, 9.8%-23.0%) vs 23/594 (3.9%; 95% CI, 1.9%-7.9%) in trap samples. Detection was clustered by ES site and correlated with site catchment population in Vellore but not Blantyre. Incidence of culture-confirmed typhoid in local hospitals was low during the study and zero some months in Vellore despite S. Typhi detection in ES. CONCLUSIONS: ES describes the prevalence and distribution of S. Typhi even in the absence of typhoid cases and could inform vaccine introduction. Expanded implementation and comparison with clinical and serological surveillance will further establish its public health utility.

Sensitive poliovirus detection using nested PCR and nanopore sequencing: a prospective validation study.

Timely detection of outbreaks is needed for poliovirus eradication, but gold standard detection in the Democratic Republic of the Congo takes 30 days (median). Direct molecular detection and nanopore sequencing (DDNS) of poliovirus in stool samples is a promising fast method. Here we report prospective testing of stool samples from suspected polio cases, and their contacts, in the Democratic Republic of the Congo between 10 August 2021 and 4 February 2022. DDNS detected polioviruses in 62/2,339 (2.7%) of samples, while gold standard combination of cell culture, quantitative PCR and Sanger sequencing detected polioviruses in 51/2,339 (2.2%) of the same samples. DDNS provided case confirmation in 7 days (median) in routine surveillance conditions. DDNS enabled confirmation of three serotype 2 circulating vaccine-derived poliovirus outbreaks 23 days (mean) earlier (range 6-30 days) than the gold standard method. The mean sequence similarity between sequences obtained by the two methods was 99.98%. Our data confirm the feasibility of implementing DDNS in a national poliovirus laboratory.

Comparison of Eleven RNA Extraction Methods for Poliovirus Direct Molecular Detection in Stool Samples.

Direct detection by PCR of poliovirus RNA in stool samples provides a rapid diagnostic and surveillance tool that can replace virus isolation by cell culture in global polio surveillance. The sensitivity of direct detection methods is likely to depend on the choice of RNA extraction method and sample volume. We report a comparative analysis of 11 nucleic acid extraction methods (7 manual and 4 semiautomated) for poliovirus molecular detection using stool samples (n = 59) that had been previously identified as poliovirus positive by cell culture. To assess the effect of RNA recovery methods, extracted RNA using each of the 11 methods was tested with a poliovirus-specific reverse transcription-quantitative PCR (RT-qPCR), a pan-poliovirus RT-PCR (near-whole-genome amplification), a pan-enterovirus RT-PCR (entire capsid region), and a nested VP1 PCR that is the basis of a direct detection method based on nanopore sequencing. We also assessed extracted RNA integrity and quantity. The overall effect of extraction method on poliovirus PCR amplification assays tested in this study was found to be statistically significant (P < 0.001), thus indicating that the choice of RNA extraction method is an important component that needs to be carefully considered for any diagnostic based on nucleic acid amplification. Performance of the methods was generally consistent across the different assays used. Of the 11 extraction methods tested, the MagMAX viral RNA isolation kit used manually or automatically was found to be the preferable method for poliovirus molecular direct detection considering performance, cost, and processing time. IMPORTANCE Poliovirus, the causative agent of poliomyelitis, is a target of global eradication led by the World Health Organization since 1988. Direct molecular detection and genomic sequencing without virus propagation in cell culture is arguably a critical tool in the final stages of polio eradication. Efficient recovery of good-quality viral RNA from stool samples is a prerequisite for direct detection by nucleic acid amplification. We tested 11 nucleic acid extraction methods to identify those facilitating sensitive, fast, simple, and cost-effective extraction, with flexibility for manual and automated protocols considered. Several different PCR assays were used to compare the recovered viral RNA to test suitability for poliovirus direct molecular detection. Our findings highlight the importance of choosing a suitable RNA extraction protocol and provide useful information to diagnostic laboratories and researchers facing the choice of RNA extraction method for direct molecular virus detection from stool.

Sustained detection of type 2 poliovirus in London sewage between February and July, 2022, by enhanced environmental surveillance.

BACKGROUND: The international spread of poliovirus exposes all countries to the risk of outbreaks and is designated a Public Health Emergency of International Concern by WHO. This risk can be exacerbated in countries using inactivated polio vaccine, which offers excellent protection against paralysis but is less effective than oral vaccine against poliovirus shedding, potentially allowing circulation without detection of paralytic cases for long periods of time. Our study investigated the molecular properties of type 2 poliovirus isolates found in sewage with an aim to detect virus transmission in the community. METHODS: We performed environmental surveillance in London, UK, testing sewage samples using WHO recommended methods that include concentration, virus isolation in cell culture, and molecular characterisation. We additionally implemented direct molecular detection and determined whole-genome sequences of every isolate using novel nanopore protocols. FINDINGS: 118 genetically linked poliovirus isolates related to the serotype 2 Sabin vaccine strain were detected in 21 of 52 sequential sewage samples collected in London between Feb 8 and July 4, 2022. Expansion of environmental surveillance sites in London helped localise transmission to several boroughs in north and east London. All isolates have lost two key attenuating mutations, are recombinants with a species C enterovirus, and an increasing proportion (20 of 118) meet the criterion for a vaccine-derived poliovirus, having six to ten nucleotide changes in the gene coding for VP1 capsid protein. INTERPRETATION: Environmental surveillance allowed early detection of poliovirus importation and circulation in London, permitting a rapid public health response, including enhanced surveillance and an inactivated polio vaccine campaign among children aged 1-9 years. Whole-genome sequences generated through nanopore sequencing established linkage of isolates and confirmed transmission of a unique recombinant poliovirus lineage that has now been detected in Israel and the USA. FUNDING: Medicines and Healthcare products Regulatory Agency, UK Health Security Agency, Bill & Melinda Gates Foundation, and National Institute for Health Research Medical Research Council.

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

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 to ensure an appropriate response. This cell culture algorithm takes 2 to 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 of our protocol compared with those of cell culture were 90.9% (95% confidence interval, 75.7% to 98.1%) and 99.2% (95.5% to 100.0%) for wild-type 1 poliovirus, 92.5% (79.6% to 98.4%) and 98.7% (95.4% to 99.8%) for vaccine and vaccine-derived serotype 2 poliovirus, and 88.3% (81.2% to 93.5%) and 93.2% (88.6% to 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 the 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.