Use of a novel real-time PCR assay to detect oral polio vaccine shedding and reversion in stool and sewage samples after a mexican national immunization day

Abstract

During replication, oral polio vaccine (OPV) can revert to neurovirulence and cause paralytic poliomyelitis. In individual vaccinees, it can acquire specific revertant point mutations, leading to vaccine-associated paralytic poliomyelitis (VAPP). With longer replication, OPV can mutate into vaccine-derived poliovirus (VDPV), which causes poliomyelitis outbreaks similar to those caused by wild poliovirus. After wild poliovirus eradication, safely phasing out vaccination will likely require global use of inactivated polio vaccine (IPV) until cessation of OPV circulation. Mexico, where children receive routine IPV but where OPV is given biannually during national immunization days (NIDs), provides a natural setting to study the duration of OPV circulation in a population primarily vaccinated with IPV. We developed a real-time PCR assay to detect and distinguish revertant and nonrevertant OPV serotype 1 (OPV-1), OPV-2, and OPV-3 from RNA extracted directly from stool and sewage. Stool samples from 124 children and 8 1-liter sewage samples from Orizaba, Veracruz, Mexico, collected 6 to 13 weeks after a NID were analyzed. Revertant OPV-1 was found in stool at 7 and 9 weeks, and nonrevertant OPV-2 and OPV-3 were found in stool from two children 10 weeks after the NID. Revertant OPV-1 and nonrevertant OPV-2 and -3 were detected in sewage at 6 and 13 weeks after the NID. Our real-time PCR assay was able to detect small amounts of OPV in both stool and sewage and to distinguish nonrevertant and revertant serotypes and demonstrated that OPV continues to circulate at least 13 weeks after a NID in a Mexican population routinely immunized with IPV.

Fig. 1.

Lower limits of detection of OPV-1, -2, and -3 nonrevertant and revertant real-time PCR assays. qPCR, quantitative (real-time) PCR. (a, c, and e) Assays for OPV serotypes 1, 2, and 3, respectively, that are nonrevertant (without the point mutation associated with vaccine-associated paralytic poliomyelitis in the 5′ untranslated region); (b, d, and f) assays for revertant OPV serotypes 1, 2, and 3, respectively. Our revertant proportion equation, from which we determine if an isolate is primarily nonrevertant or revertant, takes into account the results of both the nonrevertant and revertant assays for each serotype. Consequently, although the revertant OPV-1 assay is not very specific for revertant OPV-1 (b), given the high specificity of the nonrevertant OPV-1 assay (a), revertant versus nonrevertant OPV-1 can reliably be distinguished using the revertant proportion equation. Each point represents a serial dilution of control nonrevertant or revertant OPV of a specific serotype that was spiked into stool and subsequently underwent RNA extraction, reverse transcription, and real-time PCR. The concentration where the plotted line crosses a C_T of 35, our internal cutoff for a positive result, represents the lower limit of detection of that assay for that specific type of OPV.

Fig. 2.

Ability of the OPV-1, -2, and -3 real-time PCR assays to distinguish nonrevertant and revertant strains at different cycle thresholds. qPCR, quantitative (real-time) PCR. Revertant proportion is the proportion of virus that contains the revertant point mutation in the 5′ untranslated region that is associated with vaccine-associated paralytic poliomyelitis. A revertant proportion close to 1.0 indicates that most of the virus has the revertant point mutation, while a revertant point mutation close to 0.0 indicates that most of the virus has not developed this point mutation. Of note, a cycle threshold number above 35 is considered negative for the presence of OPV.