Kinetics of Poliovirus Shedding following Oral Vaccination as Measured by Quantitative Reverse Transcription-PCR versus Culture

Abstract

Amid polio eradication efforts, detection of oral polio vaccine (OPV) virus in stool samples can provide information about rates of mucosal immunity and allow estimation of the poliovirus reservoir. We developed a multiplex one-step quantitative reverse transcription-PCR (qRT-PCR) assay for detection of OPV Sabin strains 1, 2, and 3 directly in stool samples with an external control to normalize samples for viral quantity and compared its performance with that of viral culture. We applied the assay to samples from infants in Dhaka, Bangladesh, after the administration of trivalent OPV (tOPV) at weeks 14 and 52 of life (on days 0 [pre-OPV], ؉4, ؉11, ؉18, and ؉25 relative to vaccination). When 1,350 stool samples were tested, the sensitivity and specificity of the quantitative PCR (qPCR) assay were 89 and 91% compared with culture. A quantitative relationship between culture ؉ / qPCR ؉ and culture ؊ /qPCR ؉ stool samples was observed. The kinetics of shedding revealed by qPCR and culture were similar. qPCR quantitative cutoffs based on the day ؉11 or ؉18 stool samples could be used to identify the culture-positive shedders, as well as the long-duration or high-frequency shedders. Interestingly, qPCR revealed that a small minority (7%) of infants contributed the vast majority (93 to 100%) of the total estimated viral excretion across all subtypes at each time point. This qPCR assay for OPV can simply and quantitatively detect all three Sabin strains directly in stool samples to approximate shedding both qualitatively and quantitatively. Both immunity to paralytic poliomyelitis and reduction of transmission of wild-type and vaccine-derived polioviruses are critical in polio eradication efforts (1, 2). Oral polio vaccine (OPV) is widely utilized in low-income settings for ease of delivery, low cost, and potential for mucosal immunity. OPV immunogenicity is imperfect (3), however, and shedding of vaccine strains further complicates the poliovirus reservoir. Measurement of poliovirus in stool samples can thus be useful in evaluating mucosal immunogenicity (4), in estimating the transmission intensities of vaccine-derived virus, and for surveillance after polio eradication (5).Poliovirus culture is still considered the gold standard procedure but requires up to two 7-day passages in two cell lines, followed by molecular or antigenic methods to differentiate and type the poliovirus isolates (6). This process is time-consuming and greatly diminishes the number of stool samples that can be surveyed. Furthermore, subcultured isolates are not always representative of the viruses present in the original stool sample and results are usually not quantitated by titer determination (7,8). PCR methods are therefore attractive in terms of cost, time, the ability to subtype, and quantitation (9-12). PCR for poliovirus has been found to be severalfold more sensitive than poliovirus isolation from cell culture (13) and has also been found to be more sensitive with cerebrospinal fluid, throat swabs, or stool samples (14-17). However, s...

Comparison of culture, single and multiplex real‐time PCR for detection of Sabin poliovirus shedding in recently vaccinated Indian children

“…The RT‐PCR assay showed an increase of 12%, 18%, and 50% in the detection rates of poliovirus types 1, 2, and 3 compared to culture . Another recent study found the sensitivity and specificity of a multiplex one‐step real‐time quantitative PCR assay for the detection of Sabin poliovirus in stool samples to be 89% and 91%, respectively, compared to culture . Similar to our findings, the study also showed a significantly higher quantity of the vaccine virus detected by the multiplex PCR in culture positive samples compared to samples which were culture negative .…”

“…The multiplex one step reverse transcription real‐time PCR used to detect tOPV shedding for all three Sabin types simultaneously were performed according to Taniuchi et al The S1 and S2 probes were minor‐groove binding TaqMan probes (Applied Biosystems) with the VIC and FAM fluorophores, respectively. The S3 probe was labeled with Texas Red and BHQ2 (Integrated DNA technologies, Coralville, IA) while the MS2 probe was labeled with Quasar 670 and BHQ2 (Biosearch Technologies, Novato, CA).…”

“…The cycling conditions were 15 min at 50°C, 1 min at 95°C, and 40 cycles of 10 s at 95°C, and 45 s at 60°C on the Quantstudio 12K Flex real‐time PCR detection system (Applied Biosystems). A cutoff Ct value of 36 for S1 and 37 for S2 and S3 were applied …”

Interference of Vaccine Derived Polio Viruses with Diagnosis of Enteroviral Diseases in Neonatal Period

“…Polio virus excretion generally stopped by the end of the second week after each vaccination [11]. In Bangladesh 17.6% of the stool samples from infants were positive for polioviruses up to 25 days after vaccination [12]. This rate was 15%-16% in our study, although all the cases in our group were not in the first 2 weeks after vaccination.…”

Assessing the individual risk of fecal poliovirus shedding among vaccinated and non-vaccinated subjects following national health weeks in Mexico

“…In order to increase the specificity for OPV virus, 256 samples, including all 144 samples that were positive for at least one poliovirus serotype and 112 randomly selected negative samples using the above described rRT-PCR assays were processed using a two-step, serotype-specific multiplex rRT-PCR that was adapted from the literature [10, 11] and described in [12]. …”