EXTENDING THE UTILITY OF THE WHO RECOMMENDED ASSAY FOR DIRECT DETECTION OF ENTEROVIRUSES FROM CLINICAL SPECIMEN FOR RESOLVING POLIOVIRUS CO-INFECTION

Abstract

In a polio-free world there might be reduced funding for poliovirus surveillance. There is therefore the need to ensure that enterovirologist globally, especially those outside the global polio laboratory network (GPLN), can participate in poliovirus surveillance without neglecting their enterovirus type of interest. To accomplish this, assays are needed that allow such active participation.\n\nIn this light, we used 15 previously identified enterovirus isolates as reference samples for assay development. The first eight were enterovirus species B (EV-B). The remaining seven were EV-Cs; three of which were poliovirus (PV) 1, 2 and 3, respectively. A 16th sample was compounded; a mixture of two EV-Bs, three PVs and one nonPV EV-Cs (all part of the 15). In all, four samples contained PVs with the 16th consisting of mixture of the three PV types. All were subjected to the WHO recommended RT-snPCR assay, and five other modified (with substitution of the second round PCR forward primer) assays. The new primers included the previously described Species Resolution Primers (SRPs; 187and 189) and the Poliovirus Resolution Primers (PRPs: Sab 1, 2 and 3). All amplicons were sequenced and isolate identity confirmed using the Enterovirus Genotyping Tool.\n\nThe PRPs detected PV types in only the four samples that contained PVs. In addition, it was able to show that the sample 16 (mixture) contained all the three PV types. On the other hand, though the SRPs and the WHO assay also detected the three singleton PVs, in sample 16, they both detected only one of the three PV types present.\n\nThis study describes a sensitive and specific utility extension of the recently recommended WHO RT-snPCR assay that enables independent detection of the three poliovirus types especially in cases of co-infection. More importantly, it piggy-backs on the first round PCR product of the WHO recommended assay and consequently ensures that enterovirologists interested in nonpolio enteroviruses can continue their investigations, and contribute significantly and specifically to poliovirus surveillance, by using the excess of their first round PCR product.