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Journal of Clinical Microbiology, August 2008, p. 2671-2680, Vol. 46, No. 8
0095-1137/08/$08.00+0     doi:10.1128/JCM.00258-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Marked Variability of BK Virus Load Measurement Using Quantitative Real-Time PCR among Commonly Used Assays{triangledown}

Noah G. Hoffman,1 Linda Cook,1 Ederlyn E. Atienza,1 Ajit P. Limaye,1,2 and Keith R. Jerome1,3*

Department of Laboratory Medicine,1 Department of Medicine (Infectious Diseases), University of Washington Medical Center, Seattle, Washington 98195,2 Vaccine and Infectious Disease Institute, Fred Hutchinson Cancer Research Center, Seattle, Washington 981093

Received 7 February 2008/ Returned for modification 21 April 2008/ Accepted 9 June 2008

BK virus (BKV) is the infectious cause of polyomavirus-associated nephropathy. Screening guidelines for renal-transplant recipients define levels of viremia and viruria that are actionable for additional testing or intervention. However, standardized real-time PCR primers, probes, and standards are unavailable, and the extent of agreement among published assays is unknown. We compared seven TaqMan real-time PCR primer/probe sets (three designed at this institution, three described in the literature, and one purchased) in conjunction with two different standards to prospectively measure BKV titers in 251 urine specimens submitted to our clinical laboratory. We observed substantial disagreement among assays attributable both to features of primer and probe design and to choice of reference material. The most significant source of error among individual specimens was primer or probe mismatch due to subtype-associated polymorphisms, primarily among subtype III and IV isolates. In contrast, measurement of the most abundant subtypes (Ia, V, and VI) were typically uniform among all seven assays. Finally, we describe and validate a new clinical assay designed to reliably measure all subtypes encountered in our study population (Ia, Ic, III, IV, and VI). Consideration of available BKV sequence information in conjunction with details of subtype distribution allowed us to develop a redesigned assay with markedly improved performance. These results suggest that both accurate BKV measurement and the uniform application of BKV screening guidelines could be significantly improved by the use of standardized reference materials and PCR primers and probes.

* Corresponding author. Mailing address: Fred Hutchinson Cancer Research Center, 1100 Fairview Ave. N, D3-100 Seattle, WA 98109. Phone: (206) 667-6793. Fax: (206) 667-4411. E-mail: kjerome{at}u.washington.edu

{triangledown} Published ahead of print on 18 June 2008.

Journal of Clinical Microbiology, August 2008, p. 2671-2680, Vol. 46, No. 8
0095-1137/08/$08.00+0     doi:10.1128/JCM.00258-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Luo, C., Bueno, M., Kant, J., Martinson, J., Randhawa, P. (2009). Genotyping Schemes for Polyomavirus BK, Using Gene-Specific Phylogenetic Trees and Single Nucleotide Polymorphism Analysis. J. Virol. 83: 2285-2297 [Abstract] [Full Text]  


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