The journey of a stool sample: Understanding polio surveillance
In Pakistan and across the world, the poliovirus surveillance system and laboratory network are critical to eradication efforts.
Surveillance is one of the main pillars of the polio eradication initiative. By testing stool samples collected from children suffering acute flaccid paralysis – the clearest symptom of the virus – as well as samples taken from sewage water, we are able to find the poliovirus wherever it is hiding.
Pakistan’s polio surveillance system is one of the largest ever established in the world. Click through these pictures to learn about the journey of a stool sample there: From a child with suspected polio to the laboratory.
In Pakistan, a wide network of health workers, teachers, and other community members vigilantly look out for signs of polio in children in their area. These volunteers detect and report to the polio surveillance system children showing possible symptoms of the virus, often floppy or weakened limbs with rapid onset of paralysis, known as acute flaccid paralysis. Every suspected case acts as a signal that polio might be circulating in the area, and triggers an investigation.
The poliovirus lives in children’s intestines, where it multiplies, and is finally excreted. When a child with acute flaccid paralysis is reported to the surveillance system, health workers collect the child’s stool samples and transport them to the lab in specially designed cool boxes. The boxes ensure a constant temperature of between 4 to 8 °C, so that the viruses in the samples remain high enough quality to test. Once collected, stool samples from all corners of the country must reach the Pakistan Regional Reference Laboratory for polio eradication, based in Islamabad, within 72 hours.
The Regional Reference Laboratory was established in 1991 and tests around 30 000 stool samples each year from both Pakistan and Afghanistan. More than 99% of these stool samples come back negative for polio. This is because most cases of acute flaccid paralysis are not caused by poliovirus. However, the tiny fraction of positive results tells the programme where the virus may be hiding.
Dr Salmaan Sharif is a Molecular Biologist, and the Coordinator of the Regional Reference Laboratory in Islamabad. He supervises a team of 34 lab staff, each responsible for a different component of surveillance sample testing. With an increase in reported acute flaccid paralysis cases, and environmental sampling sites, the workload of Dr. Sharif and his team is increasing. This is a sign that the polio surveillance system is working well, as a large number of reported acute flaccid paralysis cases and environmental samples gives us our best chance of finding the virus.
In the sample receiving room, each stool sample is checked and assigned a unique laboratory identity number.
Each sample is then processed in a centrifuge, which separates the components of the sample. Solids will drop to the bottom and liquids will remain at the top. Any poliovirus will remain in the liquid component. Once separated, a scientist is ready to inoculate a healthy cell.
From the processing room onwards, extensive biosafety measures are taken to ensure that all virus is kept in the controlled environment, that contamination of other samples does not occur, and humans are not at risk of exposure. During the primary screening, healthy cells are mixed with the liquid component of the processed stool sample, and are then grown in an incubator at 36 °C for up to ten days. An incubator mimics the natural environment of the virus, creating the ideal conditions for the virus to grow. If the virus is present in the processed stool sample, it will infect the healthy cells.
A daily microscopic examination is performed to determine whether poliovirus is present in the processed stool sample. Infected cells are visibly broken in pieces, while healthy cells are seen as long strands.
If poliovirus is isolated in a stool sample, further tests are carried out to determine what type of virus this is and where the strain may have originated. A Polymerase Chain Reaction (PCR) machine is used to determine the kind of poliovirus detected (known as the serotype), and to distinguish further between wild poliovirus and that related to the vaccine-derived.
The next step is genetic sequencing. By reading the genetic code of the virus, wild viruses can be compared to others and classified into genetic families. From this, the geographic origin of the virus can be determined. This helps to guide the programme when deciding the best immunization strategies to stop transmission, and to prevent further spread of the poliovirus.
The polio surveillance system stretches even further than stool sample testing. In carefully selected places where the virus could be hiding, surveillance officers collect sewage samples. These are also sent to the Regional Reference Laboratory for processing, using a method similar to the testing of the stool samples. Crucially, environmental surveillance can help find polio in the environment before it has a chance to paralyse a child.
In Pakistan, the programme has used environmental surveillance to test and detect the presence of poliovirus in the sewage in high-risk locations since 2009. The network has continued to expand over time with a wider coverage. Currently, there are 53 sampling sites in 33 districts and towns of the country, making it the largest environmental polio surveillance network ever established.
The sensitive polio surveillance system finds the poliovirus wherever it exists – from the most remote villages to huge cities. With the polio case count at the lowest level in the history of Pakistan, the country intends to make 2018 the year of poliovirus interruption, keeping current and future generations of children safe from this disease. Thanks to the generous support of the Government of Japan, the Islamabad laboratory is continuing to expand operations, now able to procure new state-of-the-art molecular biology equipment to help detect the last remaining reservoirs of the virus.
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