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

Abstract

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.

Fig. 1. Median time required for each diagnostic step in the two protocols for 27 cVDPV2 positive stool samples.

Time taken between case onset and a sequence being generated via DDNS and the standard algorithm were compared using a two-sided Mann–Whitney U test. Source data

Fig. 2. cVDPV2 outbreaks detected by DDNS.

a, Cases from the four Maniema lineages detected during the study period (Maniema Province highlighted in green). Cases are plotted by district, with placement within the district determined at random. b, Tip-dated phylogenetic tree showing the maximum likelihood emergence date of RDC-Man-3 lineage and its subsequent diversification over time. Solid tips indicate that the DDNS detection was matched by a cell-culture-based detection of a cVDPV2 from the same sample. Cases confirmed by Sanger sequencing but without a corresponding DDNS sequence (n = 2) were not included in the analysis. Source data