NEW YORK (GenomeWeb) – Tracking immunity and virus shedding after oral polio vaccination usually requires culture of poliovirus from stool samples. Now, a team of researchers in the US and Bangladesh has developed a multiplex real-time RT-PCR method to amplify the oral vaccine's polio strains, which may ultimately help further global eradication goals.
Although many nations inoculate babies with inactivated poliovirus vaccine, oral vaccination with three attenuated Sabin strains is a widely used strategy in low-resource settings because it is cheap and easy. And while injectable vaccine does not result in good intestinal immunity, oral vaccine provides mucosal immunity in the intestines, which can end person-to-person polio transmission after mass administration campaigns, according the World Health Organization. Unfortunately, orally administered virus can sometimes be shed in human waste and transferred into the community, complicating estimates of the poliovirus reservoir.
In a study published last week in The Journal of Clinical Microbiology, a cohort of babies in Dhaka, Bangladesh, was given four doses of oral vaccine over approximately nine months. The new multiplex RT-qPCR method was compared to virus culture using 1,350 stool specimens from these infants.
Sensitivity and specificity of the multiplex assay were both around 90 percent versus culture. Interestingly, the new method also showed that some babies are "high shedders" of live polio virus after oral vaccination. Using an index which takes into account both duration of shedding and quantity of virus shed, the top three shedders accounted for more than 90 percent of the cohort's excretion.
Lead author on the study Mami Taniuchi, a scientist at the University of Virginia's Center for Global Health, said in an email to PCR Insider earlier this week that incorporation of an internal control target and use of extracted RNA from stool made the new assay particularly novel and useful.
"In low-income countries such as Bangladesh there are high incidences of fecal-oral transfer, thus it is important to have good mucosal immunity to prevent transmission of poliovirus, both wild-type and vaccine derived," Taniuchi explained. This transmission mechanism is less likely in higher-income countries, where the slight dangers associated with shed virus after oral vaccination tip the balance in favor of injected vaccine.
The study used an RNA extraction kit from Qiagen on the stool samples. The internal control accounted for extraction and amplification efficiency differences, Taniuchi said, which can be caused by PCR inhibition common with stool samples.
During development of the test, which took about three months, the group first assessed assays for each of the Sabin strains individually.
For the multiplexing reaction, they also ran head-to-head tests evaluating RT kits and mixes from Life Technologies, Takara, Qiagen, Quanta Biosciences, and Bioline, to test them "for higher sensitivity, higher specificity, lower limits of detection, [and] ease-of-use (the less reagents to mix the better)," Taniuchi said. She noted that they also assessed the “ruggedness” of the RT-PCR enzyme kits, since traveling with these enzymes in checked luggage is not uncommon, and they must be able to go through several freeze-thaw cycles and still perform well.
The study found the kit from Bioline to have the most consistent performance
For now, Taniuchi suggested the group has achieved its goal of "a simple, rapid detection of Sabin strains for use in resource-limited settings," and will continue to investigate kinetics of shedding.
As for the high shedders, she said her group hypothesizes that these infants may have impaired immunity due to malnutrition or environmental enteropathy ― a condition caused by constant fecal-oral contamination, which results in intestinal inflammation ― and are unable to mount a normal immune response to the vaccine. RT-qPCR may prove a quick and easy method to identify these high-risk shedders, Taniuchi said.