In this paper we describe our novel real-time PCR assay that can detect and accurately distinguish nonrevertant and revertant OPV serotypes 1, 2, and 3 at levels as low as or lower than 37 CCID50
virus in 100 μl of stool from RNA extracted directly from stool and sewage. Using this assay, we show that OPV continues to circulate at least 13 weeks after a NID in a Mexican community primarily vaccinated with IPV. IPV is felt to provide inferior intestinal immunity compared to OPV, and consequently, OPV circulation might continue for a longer period in this population than in a community primarily vaccinated with OPV. Children vaccinated with IPV have been found to have increased OPV shedding after an OPV challenge than children primarily vaccinated with OPV (37% IPV-vaccinated children with continued shedding at 3 weeks versus 5% of OPV-vaccinated children with continued shedding at 3 weeks) but less OPV shedding than previously unvaccinated children (of whom 81% were still shedding at 3 weeks) (2
The duration of shedding that we report is longer than that reported in a New Zealand study, which monitored stool and sewage for the presence of OPV after New Zealand changed to a national IPV vaccination regimen (5
). In New Zealand, OPV was detected in stools at up to 4 weeks and in sewage at up to 12 weeks after the change. The longer duration of shedding that we report could be due to differences in climate and hygiene between the two countries or because the cell culture method to detect OPV used in the New Zealand study may have been less sensitive at detecting OPV than our PCR method. Our results appear to be similar to those from a study in Cuba, where IPV is not used and polio vaccination is done exclusively through semiannual NIDs with OPV (13
). The Cuban study, using a cell culture technique to detect OPV, found that OPV was detectable in stool samples until between 7 and 15 weeks and in sewage until between 15 and 19 weeks after an NID. The proportions of children with detectable OPV in stool samples in the Cuban study were approximately 25% 3 to 6 weeks after vaccination, 13% by 7 weeks after vaccination, and 0% by 15 weeks after vaccination. However, since we did not continue the pilot study beyond 13 weeks, the duration of OPV circulation in the community that we studied in Mexico may extend beyond what we report. Indeed, in our study, sewage was collected during the rainy season and was visibly more dilute than at other times during the year, yet OPV was still detectable. This suggests that because OPV shedding was still prevalent at 13 weeks under such conditions, shedding may have continued for some weeks further. We are currently conducting a larger, longitudinal study in the same Mexican community to determine the precise duration of OPV circulation.
A surprising result of our study was that the OPV-2 and -3 detected in both stool and sewage 6 to 13 weeks after the NID were predominantly nonrevertant. This conflicts with historical data, from both Mexico and elsewhere, in which by 2 weeks after receipt of OPV, almost all of the OPV shed by vaccinees is revertant with the characteristic point mutations in the 5′ untranslated region (4
). The etiology of this discrepancy is unclear. Mexico has recently changed from propagation of OPV seed strains in primary monkey kidney cells to Vero cells (16
). However, there are no convincing data that propagation in Vero cells increases the stability of OPV, and indeed, one study showed no modification in the pattern of poliovirus excretion when children vaccinated with OPV propagated in Vero cells were compared with children vaccinated with OPV propagated in primary monkey kidney cells (11
). We were unable to confirm the absence of the point mutations by sequencing our samples, due to the low concentrations of virus present. However, our real-time PCR assay detected primarily revertant OPV, as expected, from stool samples from a separate study in Africa looking at OPV shedding (data not shown). We were able to sequence some of these isolates from African stool samples that had high OPV concentrations, and in all cases the sequence results corresponded to our real-time PCR results in terms of the presence or absence of the revertant point mutations. This suggests that the etiology of the absence of revertant OPV-2 and OPV-3 is not due to a flaw in our assay but rather is inherent in the samples themselves.
One limitation of our study is that the conditions in the Mexican community approximate, but do not replicate, what might occur if wild poliovirus is eradicated and there is global OPV cessation and initiation of IPV. In the latter scenario, moving forward from the date of OPV cessation, any child too young to have been vaccinated would be vaccinated only with IPV. In the Mexican community that we studied, while all the children had received IPV as their primary regimen, 45% of them had also received OPV during a national immunization day. Consequently, they might have higher intestinal immunity than children vaccinated solely with IPV. In addition, while the environmental conditions in Mexico are similar to those in countries that most favor poliovirus spread, Mexico has very high vaccination rates. Consequently, Mexico is not 1 of the 16 countries, primarily located in Africa and southern Asia, that suffered an outbreak of VDPV in the last decade (18
). These are countries where OPV circulation after global OPV cessation would be expected to continue the longest. However, until these countries implement a national change from OPV to IPV, the unique vaccination policy in Mexico best allows us to study OPV community circulation in a country that has switched to using IPV as its primary vaccination regimen.
In summary, our real-time PCR assay was able to detect very low concentrations of OPV in both stool and sewage and to distinguish nonrevertant and revertant serotypes. Using this assay, we were able to determine that OPV circulation continues for at least 13 weeks after a NID in a Mexican community primarily vaccinated with IPV. Further studies are ongoing to better define the duration of OPV circulation and mutation after a NID in Mexico.