Tackling poliovirus outbreaks and eliminating cell-culture detection methods with nanopore technology

The battle to completely eradicate poliovirus

Poliomyelitis (polio) is a viral disease transmitted via faecal-oral contamination and spreads rapidly in areas with poor sanitation. This infectious disease has varied clinical features, often starting with flu-like symptoms, leading to severe muscle spasms. However, its hallmark in children is flaccid paralysis of the limbs that can be permanent, and in extreme cases, respiratory paralysis that can cause death¹. Polio was a global disease until the first successful large-scale poliovirus vaccine trials in 1954 in the USA, which led to widespread use of the vaccine in developed countries, significantly reducing outbreaks². In 1988, the Global Polio Eradication Initiative (GPEI) was launched to further tackle this harmful disease, resulting in wild poliovirus being almost eradicated in all countries, excluding Afghanistan and Pakistan³.

However, poliovirus is still a public health concern in countries with low vaccine coverage and, as a result, the GPEI has rolled out mass vaccination campaigns using the oral poliovirus vaccine (OPV) ⁴. The live, weakened virus in the OPV spreads within a population to widen immunisation; but prolonged circulation can lead to the attenuating mutations in the weakened virus to reverse in under-vaccinated populations, resulting in outbreaks of neurovirulent circulating vaccine-derived poliovirus (cVDPV)^4,5.

Detecting and confirming outbreaks is slow and complex

The Democratic Republic of the Congo (DRC) has continued to have cases of cVDPV since their last incidence of wild poliovirus 10 years ago⁴. To prevent the spread of poliovirus in the DRC, all reports of acute flaccid paralysis are investigated by collecting stool samples from the affected individual, and their close contacts, within two weeks of the onset of paralysis^4,6. The samples are then sent to the only World Health Organization (WHO)-accredited laboratory in the DRC, which performs cell culture and intratypic differentiation (ITD) quantitative PCR to initially detect poliovirus. To confirm the presence of poliovirus and to distinguish strains, positive samples must then be sent to a laboratory in South Africa for Sanger sequencing of the VP1 capsid region⁴.

This cell-culture-based process is slow and complex, increasing the time to results and leading to delays in outbreak detection and response^4,6. For each additional week between paralysis onset and vaccination, outbreak cases are estimated to increase by 12%⁶. To effectively combat polio endemics, outbreaks must be confirmed faster to prevent delays in treatment and to limit outbreak size and duration by implementing appropriate response plans.

Overcoming logistical challenges with nanopore sequencing

The WHO Polio Eradication Strategy 2022–2026 aims to remove cell-culture-based detection methods and implement country-level direct detection strategies to speed up poliovirus investigations⁷. Direct molecular detection and nanopore sequencing (DDNS) offers the possibility to detect poliovirus outbreaks locally⁸. DDNS combines fast, simple library preparation, which requires minimal laboratory equipment, with onsite sequencing and analysis using benchtop nanopore sequencing devices to generate data in real time, ‘avoiding international transport of samples and enabling quick response to outbreaks’⁴.

To evaluate the DDNS method for poliovirus surveillance, Shaw et al. compared it with the current gold-standard culture-based method for over 2,300 stool research samples⁴. For DDNS, RNA was extracted before nested, barcoded PCR was performed, and the resulting amplicons sequenced on a GridION for 4–12 hours. The culture-based method followed the standard accredited process, including shipping poliovirus-positive samples to South Africa for Sanger sequencing.

DDNS provided results in an average of seven days and was able to confirm the serotype of the cVDPV outbreak. This was 23 days faster than the average turnaround time for the culture-based method, which included approximately eight days for shipping samples between countries (Figure 1). DDNS also offered highly accurate results, comparable to the culture-based method. However, interestingly the culture-based method produced nine false-negative results.

Figure 1. Median time required for each step in both methods for 27 cVDPV-positive stool samples. Figure from Shaw et al.⁴ and available under Creative Commons license (creativecommons.org/licenses/by/4.0).

Furthermore, multiple viral serotypes, such as Sabin-1 and Sabin-3, from a single sample were identified with DDNS due to the improved haplotype calling, meaning viruses of importance to the eradication strategy — whether wild-type or vaccine-derived — can be detected and a poliovirus outbreak confirmed with a single workflow. With the current gold-standard method, this information can only be determined when samples are shipped to a separate laboratory for Sanger sequencing.

'Direct molecular detection and nanopore sequencing (DDNS) of poliovirus in stool samples is a promising fast method.'

Increasing local sequencing capacity to improve disease surveillance

Shaw et al. confirmed that ‘DDNS can be applied as a rapid, sensitive, and cost-effective tool for surveillance in the DRC’ without the need for time-consuming cell culture and is feasible to implement in national laboratories due to its simple workflow, minimal infrastructure requirements, and low cost per sample for high-throughput experiments.

The authors also suggested that the DDNS method could be repurposed to detect other pathogens locally during public health emergencies because the same facilities and sequencing equipment can be used. By training more laboratories to perform nanopore sequencing, there will be increased ‘opportunities to foster the development of these skills and facilitate their contribution to disease surveillance and pathogen genomics’.

‘The skills and facilities required for DDNS can be rapidly redeployed to other pathogens during public health emergencies.'

Finally, Shaw et al. concluded that the research team are optimising the DDNS method to also analyse environmental samples from wastewater, with the aim of generating a similar degree of detection sensitivity for continuous surveillance of poliovirus. The researchers are working to meet the requirements for the WHO Global Polio Laboratory Network recommendation, with hopes for DDNS to be used to inform poliovirus outbreak responses in the future.