RNA extraction prior to HIV-1 resistance detection using Line Probe Assay (LiPA):: comparison of three methods

Background: Genotyping testing has been accepted as a guidance in the therapeutic management of Human Immunodeficiency virus 1 (HIV-1). However, optimization of the available routine techniques for such purpose has not been fulfilled. Objective: To evaluate the use of three RNA extraction methods in order to be applied in the genotypic HIV-1 resistance testing by LiPA. Study design: Comparative prospective study of three HIV-1 RNA extraction methods. Forty-eight plasma samples were tested for the determination of viral load (VL) by means of Cobas Amplicor HIV-1 Monitor (TM) (Roche Diagnostics. Branchburg, NJ, USA), preserving the obtained RNA extracts. RNA was also extracted using two other techniques: "SV Total RNA Isolation System" (Promega Corporation. Madison, WI, USA) and "QIAamp Viral RNA" (QIAGEN Inc., Valencia, CA, USA). The three RNA extracts were processed in parallel for the detection of HIV resistance by LiPA, and bands were recorded comparatively. Results: Results obtained by Roche extraction method were superior, followed by those of Qiagen and Promega, in the several studied parameters. First, proportion of amplified samples (75.0% by Promega versus 95.8 by Qiagen and 97.9% by Roche for LiPA RT and 97.7% by Promega versus 100.0% by Roche and Qiagen for LiPA P); second, percentage of combined mutations patterns, and third, differences in band intensity. Thus, for LiPA RT 51.4% and 54.3% of the samples showed greater intensity after Roche and Qiagen extractions, respectively. These percentages dropped to 12.8 and 19.1 for LiPA P. Conclusions: The outcome obtained by LiPA after RNA extraction by Roche methodology was remarkably superior to those of Promega. and Qiagen. LiPA technique needs further optimization, especially the sample amplification phase of LiPA RT. (c) 2004 Elsevier B.V. All rights reserved.