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Journal of Clinical Microbiology, August 2007, p. 2419-2425, Vol. 45, No. 8
0095-1137/07/$08.00+0     doi:10.1128/JCM.02268-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

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{triangledown}

Devasena Gnanashanmugam, Meira S. Falkovitz-Halpern, Anthony Dodge,{dagger} Melanie Fang,{ddagger} Lisa J. Wong,§ Melissa Esparza, Rebecca Hammon, Enrique E. Rivas-Merelles,|| Jose I. Santos,{dagger}{dagger} 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.

* Corresponding author. Mailing address: Department of Pediatrics, Stanford University School of Medicine, 300 Pasteur Drive, Stanford, CA 94305. Phone: (650) 723-5682. Fax: (650) 725-8040. E-mail: bonniem{at}stanford.edu

{triangledown} Published ahead of print on 20 June 2007.

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

{ddagger} 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.

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

Journal of Clinical Microbiology, August 2007, p. 2419-2425, Vol. 45, No. 8
0095-1137/07/$08.00+0     doi:10.1128/JCM.02268-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.





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