Time Taken to Detect and Respond to Polio Outbreaks in Africa and the Potential Impact of Direct Molecular Detection and Nanopore Sequencing

Abstract

Background: Detection of poliovirus outbreaks relies on a complex laboratory algorithm of cell-culture, polymerase chain reaction (PCR), and sequencing to distinguish wild-type and vaccine-derived polioviruses (VDPV) from Sabin-like strains. We investigated the potential for direct molecular detection and nanopore sequencing (DDNS) to accelerate poliovirus detection.

Methods: We analyzed laboratory data for time required to analyze and sequence serotype-2 VDPV (VDPV2) in stool collected from children with acute flaccid paralysis in Africa (May 2016-February 2020). Impact of delayed detection on VDPV2 outbreak size was assessed through negative binomial regression.

Results: VDPV2 confirmation in 525 stools required a median of 49 days from paralysis onset (10th-90th percentile, 29-74), comprising collection and transport (median, 16 days), cell-culture (7 days), intratypic differentiation quantitative reverse transcription PCR (3 days), and sequencing, including shipping if required (15 days). New VDPV2 outbreaks were confirmed a median of 35 days (27-60) after paralysis onset, which we estimate could be reduced to 16 days by DDNS (9-37). Because longer delays in confirmation and response were positively associated with more cases (P < .001), we estimate that DDNS could reduce the number of VDPV2 cases before a response by 28% (95% credible interval, 12%-42%).

Conclusions: DDNS could accelerate poliovirus outbreak response, reducing their size and the cost of eradication.

Keywords: AFP; VDPV; direct detection; nanopore; outbreak; poliovirus; stool; surveillance.

Figure 1.

Data availability for keys steps in the current method of poliovirus detection from stool samples. Cell culture result, ITD result, and sequencing results are the 3 potential end points of testing, with only a subset of samples requiring ITD (those demonstrating a cytotoxic effect of L20B cells, indicating the presence of poliovirus [L20B +ve]), and a subset of those requiring sequencing (where ITD indicates a wild-type virus, serotype 2, or a non-Sabin serotype 1 or 3). Within each end point is shown the number of samples that had dates in chronological order for all the steps required to reach that end point. Abbreviations: AFP, acute flaccid paralysis; GPLN, Global Polio Laboratory Network; ITD, intratypic differentiation; PCR, polymerase chain reaction.

Figure 2.

Time between steps in sample processing from AFP onset to cell culture or ITD results. The median time from AFP onset to (A) a cell culture result or (B) an ITD result is shown by country. The distribution of times taken for each step until (C) a cell culture result or (D) an ITD result is shown for all samples. A and B, Number above each bar indicates the number of samples originating from the country that were included in the analysis. Countries are grouped according to whether samples undergo cell culture and ITD within the country of origin or are shipped internationally for testing. Abbreviations: AFP, acute flaccid paralysis; GPLN, Global Polio Laboratory Network; ITD, intratypic differentiation.

Figure 3.

Time between steps in sample processing from AFP onset to a sequencing result for VDPV2-positive samples. A, Median time by country with the number above each bar indicating the number of samples originating from the country that were included in the analysis. Countries are grouped according to whether samples undergo cell culture and ITD within the country of origin or are shipped internationally for testing. B, Distribution of the time taken for each step and overall across all countries and samples. Abbreviations: AFP, acute flaccid paralysis; CAR, Central African Republic; DRC, Democratic Republic of the Congo; GPLN, Global Polio Laboratory Network; ITD, intratypic differentiation; VDPV2, serotype-2 vaccine-derived poliovirus.

Figure 4.

The potential impact of direct detection and poliovirus nanopore sequencing. Median intervals between AFP onset and (A) VDPV2-positive sequencing result and (B) ITD result by country. Observed intervals for the current methodology are compared with estimated intervals for the fastest possible implementation of cell culture, ITD, and sequencing, and for nanopore testing available only in GPLN laboratories or in all countries. Abbreviations: AFP, acute flaccid paralysis; CAR, Central African Republic; DRC, Democratic Republic of the Congo; GPLN, Global Polio Laboratory Network; ITD, intratypic differentiation; VDPV2, serotype-2 vaccine-derived poliovirus.

Figure 5.

Predicted impact of direct detection and nanopore sequencing on the timeliness of detection of VDPV2 outbreaks. A, Observed number of days between the earliest onset of AFP and detection of each VDPV2 outbreak within a country according to current detection methods (filled circle and grey line) and the estimated number of days for this interval across outbreaks using the nanopore method with 9% sensitivity per sample (boxplots with 90% range, black line). Boxplot ranges are often narrow as an initial AFP case with 2 collected stool samples gives a 99% probability of outbreak detection. Y-axis indicates the country of the outbreak (World Health Organization acronym code) followed by the VDPV2 lineage. B, Distribution across all country outbreaks of the interval between AFP onset and confirmation by sequencing of each VDPV2 outbreak for the 2 detection methods. Abbreviations: AFP, acute flaccid paralysis; ANG, Angola; BEN, Benin; CAE, Cameroon; CAF, Central African Republic; CHA, Chad; COD, Democratic Republic of the Congo; ETH, Ethiopia; GHA, Ghana; MOZ, Mozambique; NIE, Nigeria; NIG, Niger; TOG, Togo; VDPV2, serotype-2 vaccine-derived poliovirus; ZAM, Zambia.

Figure 6.

A, Interval in weeks between onset of AFP or environmental sample collection and notification to WHO headquarters (red) and between notification and vaccination response campaign (blue) for each outbreak in a given country. For outbreaks where vaccination took place before notification (because of a response to a nearby outbreak), overall interval between onset and response shown (green). Y-axis indicates the country of the outbreak (World Health Organization acronym code) followed by the VDPV2 lineage. Asterisks indicate outbreaks that were first detected by environmental surveillance. B, Number of VDPV2 cases between first isolate and 30 days after first response by interval between onset and response. Lines show mean expected cases, with 2.5th and 97.5th percentiles shown by ribbon. The y-axis is plotted on a pseudo-log scale. Abbreviations: AFP, acute flaccid paralysis; ANG, Angola; BEN, Benin; CAE, Cameroon; CAF, Central African Republic; CHA, Chad; COD, Democratic Republic of the Congo; ETH, Ethiopia; GHA, Ghana; MOZ, Mozambique; NIE, Nigeria; NIG, Niger; TOG, Togo; VDPV2, serotype-2 vaccine-derived poliovirus; ZAM, Zambia.