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Shedding and Reversion of Oral Polio Vaccine Type 3 in Mexican Vaccinees: Comparison of Mutant Analysis by PCR and Enzyme Cleavage to a Real-Time PCR Assay

Devasena Gnanashanmugam, Meira S. Falkovitz-Halpern, Anthony Dodge, Melanie Fang, Lisa J. Wong, Melissa Esparza, Rebecca Hammon,^¶ Enrique E. Rivas-Merelles,^|| Jose I. Santos, and Yvonne Maldonado^*

Stanford University School of Medicine, 300 Pasteur Drive, Stanford, California 94305

Received 7 November 2006/ Returned for modification 4 April 2007/ Accepted 10 June 2007

A uracil-to-cytosine mutation at nucleotide position 472 of oral poliovirus vaccine type 3 (OPV3) contributes to the development of vaccine-associated paralytic poliomyelitis (VAPP). To analyze OPV3 shedding patterns, we previously used the multistep method of mutant analysis by PCR and enzyme cleavage (MAPREC). This involves conventional reverse transcription-PCR to detect OPV3, followed by a restriction digest to quantify position 472 reversion. Real-time PCR detects and quantifies nucleic acid as PCR occurs and avoids postreaction processing. The goal of this study was to compare a real-time PCR method to MAPREC. Seventy-three stool samples from Mexican OPV recipients underwent the reverse transcription-PCR step of MAPREC and real-time PCR. Real-time PCR identified 23% more OPV3-positive samples than conventional reverse transcription-PCR. When reversion was compared, the revertant proportion (RP), defined as the percentage of revertants in a sample, differed by 10% in 21/25 (84%) samples. The four samples differing by >10% were obtained within 5 days of OPV administration. The real-time PCR assay identified samples with an RP of 85% with 94% sensitivity and 86% specificity compared to MAPREC. The mean difference in RP between the two methods was 3.6% (95% confidence interval, –0.3 to 7.5%). Real-time PCR methods reliably detect OPV3, and reversion estimates correlate more consistently with MAPREC when OPV3 reversion rates are high. Detecting VAPP-related mutations by real-time PCR is rapid and efficient and can be useful in monitoring ongoing global polio eradication efforts.

Published ahead of print on 20 June 2007.

Present address: 850 Lincoln Center Drive, Foster City, CA 94404.

Present address: 34680 Loreal Terrace, Fremont, CA 94555.

Present address: 916 Homestake Dr., Golden, CO 80401-1772.

^¶ Present address: David Geffen School of Medicine at UCLA, 765 Weyburn Place #311, Los Angeles, CA 90024.

^|| Present address: Hospital Regional de Rio Blanco, Entronque S.N. Carretera Orizaba-Puebla, Congregacion Vincente Guerrero, Rio Blanco, Veracruz. CP 94735 Mexico.

Present address: Hospital Infantil de Mexico, Federico Gómez, Dr. Marquez #162, Colonia Doctores, Mexico 06720 D.F., Mexico.