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Journal of Clinical Microbiology, March 2008, p. 1104-1105, Vol. 46, No. 3
0095-1137/08/$08.00+0     doi:10.1128/JCM.02259-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Comparison of Results Obtained with Amplicor HIV-1 DNA PCR Test Version 1.5 Using 100 versus 500 Microliters of Whole Blood{triangledown}

Estelle Piwowar-Manning,1,2 Lebah Lugalia,1,2 Bosco Kafufu,2 and J. Brooks Jackson1,2*

Department of Pathology, Johns Hopkins Medical Institutions, 600 North Wolfe Street, Baltimore, Maryland 21287,1 Makerere University-Johns Hopkins University Research Collaboration Laboratory, Old Mulago Hill Road, Kampala, Uganda2

Received 21 November 2007/ Returned for modification 19 December 2007/ Accepted 4 January 2008


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ABSTRACT
 
The Amplicor HIV-1 DNA PCR assay (Roche Diagnostics, Branchburg, NJ) requires 500 µl of whole blood for a diagnosis of human immunodeficiency virus type 1 (HIV-1) infection, and this amount is often difficult to obtain from infants. A comparison was performed using 100 and 500 µl of whole blood from infants less than 18 months of age. The concordance rate for HIV DNA PCR-negative and -positive samples was 100% for the two different volumes.


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TEXT
 
The Amplicor HIV-1 DNA PCR assay (Roche Diagnostics, Branchburg, NJ) has been used in clinical trials and in clinical practice for the diagnosis of human immunodeficiency virus (HIV) infection in infants less than 18 months of age who were born to HIV-infected mothers (1). In May 2005, Roche Diagnostics replaced the version 1.0 Amplicor HIV-1 DNA PCR assay with the version 1.5 assay, which has been shown to have excellent sensitivity and specificity in testing adult venous blood samples and infant dried blood spots (2, 3). Unlike the version 1.0 package insert, the manufacturer's version 1.5 package insert does not address the issue of testing infant samples with respect to the volume of blood that can be used (Amplicor HIV-1 DNA test version 1.5 package insert; Roche Molecular Systems, Inc., Branchburg, NJ).

The Roche Amplicor HIV-1 DNA test version 1.0 required a 100-µl blood volume from infants less than 18 months of age and 500 µl from infants greater than 18 months of age. However, according to the manufacturer's new version 1.5 package insert, 500 µl of blood is required regardless of age. As it is often difficult to obtain 500 µl of blood or more from infants who may require multiple tests on a small volume of blood, the objective of this study was to validate the use of 100 µl of blood for infants under 18 months of age in the version 1.5 assay by comparing the concordance rates of positivity and negativity for the two different blood volumes.

In this study, blood samples were obtained from 38 babies previously determined by PCR (using 500 µl of whole blood) to be positive for HIV type 1 (HIV-1) DNA and 16 HIV-1 DNA PCR-negative babies aged 6 weeks to 18 months who visited the clinics at Mulago Hospital, Kampala, Uganda, and whose mothers or guardians gave consent for HIV DNA testing. Approximately 1 ml of blood from each baby was collected in EDTA anticoagulant and transported to the Makerere University-Johns Hopkins University Uganda Core Laboratory. The processing technician at the core laboratory made one or more cell pellets from 100- and 500-µl aliquots of each blood sample (Table 1). Three milliliters of specimen wash buffer was used to prepare and wash each cell pellet from 100 or 500 µl of whole blood. The Amplicor HIV-1 DNA PCR test version 1.5 was run on the two pellets from each specimen according to the manufacturer's instructions. Kit controls and coded control pellets were used for the validation of each assay run. The technologist running the assay was blinded as to the HIV status of each infant.


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  		TABLE 1. HIV-1 DNA PCR results for testing of 100- and 500-µl samples of whole blood from infants 6 weeks to 18 months of age born to HIV-infected mothers

The concordance rate was 100% for the 16 HIV DNA PCR-negative samples and 100% for the 38 HIV DNA PCR-positive samples (Table 1). The optical density (OD) readings for the tested pellets made from 100- and 500-µl aliquots of samples from 54 different infants were not significantly different (Table 1). The mean OD of the HIV-1 DNA-positive samples made from 100 µl of whole blood was 3.91, compared with a mean OD of 3.96 for those made from 500 µl. The mean OD of the HIV-1 DNA-negative samples made from 100 µl of whole blood was 0.062, compared with a mean OD of 0.056 for those made from 500 µl.

Our comparison revealed no significant difference in the HIV-1 DNA PCR test results obtained using 100- and 500-µl blood volumes in the cell pellet preparation for the Roche Amplicor HIV-1 DNA PCR test version 1.5. A limitation of our study is that blood samples from infants less than 6 weeks of age, when HIV DNA levels may be lower, although lymphocyte counts are typically higher in very young infants, were not available for testing. Nevertheless, because of the difficulties encountered during venipuncture in infants less than 18 months old and the need to minimize the risk of anemia, our data indicate that a volume of 100 µl of blood can be used in the cell pellet preparation for the Amplicor HIV-1 DNA PCR test version 1.5, at least for infants greater than 8 weeks of age.


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ACKNOWLEDGMENTS
 
No sponsored support was received for this project. J. Brooks Jackson has received honoraria in the past year from Roche Diagnostics for two scientific presentations he gave at Roche-sponsored scientific meetings, but not on this topic.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Pathology, Carnegie 415, 600 North Wolfe St., Johns Hopkins Medical Institutions, Baltimore, MD 21287. Phone: (410) 614-4966. Fax: (410) 614-2907. E-mail: bjackso{at}jhmi.edu Back

{triangledown} Published ahead of print on 16 January 2008. Back


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REFERENCES
 
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  1. Creek, T. L., G. G. Sherman, J. Nkengasong, L. Lu, T. Finkbeiner, M. G. Fowler, E. Rivadeneira, and N. Shaffer. 2007. Infant human immunodeficiency virus diagnosis in resource-limited settings: issues, technologies, and country experiences. Am. J. Obstet. Gynecol. 197:S64-S71.[Medline]
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  2. Germer, J. J., T. M. Gerads, J. N. Mandrekar, P. S. Mitchell, and J. D. C. Yao. 2006. Detection of HIV-1 DNA proviral DNA with the AMPLICOR HIV-1 DNA test, version 1.5, following sample processing by the MagNa Pure LC instrument. J. Clin. Virol. 37:195-198.[CrossRef][Medline]
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  3. Patton, J. C., E. Akkers, A. H. Coovadia, T. M. Meyers, W. S. Stevens, and G. S. Sherman. 2007. Evaluation of dried whole blood spots obtained by heel or finger stick as an alternative to venous blood for diagnosis of human immunodeficiency virus type 1 infection in vertically exposed infants in the routine diagnostic laboratory. Clin.Vaccine Immunol. 14:201-203.

Journal of Clinical Microbiology, March 2008, p. 1104-1105, Vol. 46, No. 3
0095-1137/08/$08.00+0     doi:10.1128/JCM.02259-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.




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