Direct detection of Sabin poliovirus vaccine strains in stool specimens of first-dose vaccinees by a sensitive reverse transcription-PCR method

Abstract

A multiplex reverse transcription-PCR method was optimized to monitor the duration of excretion of Sabin poliovirus strains in stools of vaccinees following administration of the first dose of the trivalent oral vaccine. The assay detected approximately 1 50% tissue culture infective dose of each poliovirus serotype spiked into cell culture media. Although PCR inhibitors were frequently encountered in the stool specimens, a 1:20 dilution of the extracted RNA was sufficient to obtain a positive PCR result. Analysis of 195 stool specimens collected from 26 vaccinees showed that poliovirus types 1, 2, and 3 were identified more frequently by PCR than by tissue culture isolation. The percentages of specimens positive by PCR for poliovirus types 1, 2, and 3 were 67.2, 82.6, and 53.8, respectively. In contrast, the culture method identified types 1, 2, and 3 virus in 55.4, 64.1, and 27.7% of the samples, respectively. Poliovirus type 2 excretion was detected by PCR in practically all of the oral poliovirus vaccine recipients for 4 to 8 weeks following vaccination. In contrast, excretion of type 1 and 3 viruses was more variable, with a range of 1 to 8 weeks. Shedding of type 3 virus ceased in approximately 70% of vaccinees within a week after immunization. In addition to an enhanced sensitivity for the detection of poliovirus, this PCR method permits the direct characterization of virus in stool specimens without further passage in culture, which may select for genetic variants that may not accurately reflect the virus composition in the original specimen.

FIG. 1

Recovery of Sabin virus RT-PCR signals by dilution of an inhibitory extract of stool from vaccinee M. PV PCR products, indicated by arrowheads, were detected by OH assay with a cocktail of ³²P-labeled Sabin serotype-specific probes (A) and for a subset of samples from panel A by ethidium bromide staining of products electrophoresed in a 15% polyacrylamide gel (B). (A and B) Lanes: 1, Undiluted stool; 2, 1:5 dilution; 3, 1:10 dilution; 4, 1:20 dilution; 5, 1:40 dilution. (A) Lanes: NC, no RNA template negative control; 6, stool diluted 1:80; 7, no stool extract; 8, positive control extract from OPV vaccinee W. Arrows identify unhybridized probes at the bottom of panel A.

FIG. 2

Recovery of PV2 hybridization signals from inhibitory stool extracts by decreasing the S2-P probe annealing temperature in the OH assay from 62 to 57°C. Inhibitory undiluted extracts of stool specimens from OPV vaccinees, along with extracts that did not interfere with RT-PCR, were amplified, and the PCR products were detected by the OH assay with the PV2-specific probe S2-P at 57°C (lanes 1 to 5) and 62°C (lanes 6 to 10). A 30-min autoradiograph is shown. The PV2 PCR product is indicated by the arrowhead. Lanes 1 and 6, inhibitory extract of stool from vaccinee M; lanes 2 and 7, inhibitory extract of stool from vaccinee 34; lanes 3 and 8, noninhibitory extract of stool from vaccinee W; lanes 4 and 9, inhibitory extract of stool prepared from prevaccination stool (day 0) from vaccinee M spiked with Sabin PV1, PV2, and PV3; lanes 5 and 10, extract prepared from Sabin PV2 spiked in culture medium (no stool).

FIG. 3

Duration of PV shedding from an OPV recipient (vaccinee Z). Stool samples were collected before OPV administration (day 0) and on various days (days 1, 2, 3, 4, 7, 14, 21, and 28) within 1 month after OPV administration. Undiluted stool extracts were amplified, and PCR products were detected by OH assay with either a mixture of all three Sabin serotype-specific ³²P-labeled probes (lanes 1 to 7 and 32) or individual probes S1-P (lanes 8 to 15), S2-P (lanes 16 to 23), and S3-PT (lanes 24 to 31). A 2-h autoradiograph is shown. The PV PCR products are indicated by arrowheads. Lanes: 1 to 3, PCR-negative controls minus RNA template; 4, extraction-negative control; 5, sample from day 0, 6, PCR-positive control; 7, PCR-positive control spiked into an extract of a stool obtained on day 0; 8, 16, and 24, sample from day 1; 9, 17, and 25, samples from day 2; 10, 18, and 26, sample from day 3; 11, 19, and 27, sample from day 4; 12, 20, 28, sample from day 7; 13, 21, and 29, sample from day 14; 14, 22, and 30, sample from day 21; lanes 15, 23, and 31, sample from day 28; 32, extraction-positive control.