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Journal of Clinical Microbiology, September 2006, p. 3105-3113, Vol. 44, No. 9
0095-1137/06/$08.00+0     doi:10.1128/JCM.02663-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Effect of Primer Selection on Estimates of GB Virus C (GBV-C) Prevalence and Response to Antiretroviral Therapy for Optimal Testing for GBV-C Viremia

I. E. Souza,1,2 J. B. Allen,1 J. Xiang,1 D. Klinzman,1 R. Diaz,2 S. Zhang,3 K. Chaloner,3 D. Zdunek,4 G. Hess,5 C. F. Williams,6 L. Benning,7 and J. T. Stapleton1*

Department of Internal Medicine, University of Iowa Roy and Lucille Carver College of Medicine, and Iowa City Veterans Administration Medical Center, Iowa City, Iowa,1 Infectious Diseases Division, Paulista School of Medicine, Federal University of Sao Paulo, Sao Paulo, Brazil,2 Department of Biostatistics, University of Iowa College of Public Health, Iowa City, Iowa,3 Roche Diagnostics GmbH, Penzburg, Germany,4 Roche Diagnostics GmbH, Mannheim, Germany,5 Epidemiology Branch, Division of AIDS, National Institutes of Allergy and Infectious Diseases, National Institutes of Health, Department of Health and Human Services, Bethesda, Maryland,6 Johns Hopkins University, Bloomberg School of Public Health, Baltimore, Maryland7

Received 22 December 2005/ Returned for modification 3 April 2006/ Accepted 20 June 2006

GB virus C (GBV-C; also called hepatitis G virus) is a common cause of infection associated with prolonged survival among HIV-infected individuals. The prevalences of GBV-C viremia vary widely in different studies, and there has been poor agreement among different laboratories performing GBV-C RNA detection in quality control studies. To determine the optimal method of measuring GBV-C RNA in clinical samples, samples obtained from 939 HIV-infected subjects were studied using reverse transcription (RT)-PCR methods amplifying four separate regions of the GBV-C genome. Primers amplifying the E2 coding region were 100% specific; however, their sensitivity was only 76.6%. In contrast, primers amplifying three additional conserved regions of the GBV-C genome (the 5' nontranslated region and the nonstructural protein-coding regions 3 and 5A) were more sensitive but produced higher rates of false-positive results. Using low-specificity primer sets influenced the significance of association between GBV-C viremia and response to antiretroviral therapy. Using a quantitative GBV-C RNA method, the GBV-C RNA concentration did not correlate with baseline or set point HIV RNA levels; however, a correlation between negative, low, and high GBV-C RNA levels and increasing reduction in HIV RNA following antiretroviral therapy was observed. Subjects with both GBV-C E2 antibody and viremia had significantly lower GBV-C RNA levels than did viremic subjects without E2 antibody. These studies demonstrate that accurate detection of GBV-C RNA by nested RT-PCR requires the use of primers representing multiple genome regions. Analyses based on testing with single primers do not lead to reliable conclusions about the association between GBV-C infection and clinical outcomes.

* Corresponding author. Mailing address: Department of Internal Medicine, SW54-15, GH, 200 Hawkins Drive, University of Iowa, Iowa City, IA 52242. Phone: (319) 356-3168. Fax: (319) 356-4600. E-mail: jack-stapleton{at}uiowa.edu.

Journal of Clinical Microbiology, September 2006, p. 3105-3113, Vol. 44, No. 9
0095-1137/06/$08.00+0     doi:10.1128/JCM.02663-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.





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