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Journal of Clinical Microbiology, April 2009, p. 1107-1118, Vol. 47, No. 4
0095-1137/09/$08.00+0     doi:10.1128/JCM.02255-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Evaluation of Different RNA Extraction Methods and Storage Conditions of Dried Plasma or Blood Spots for Human Immunodeficiency Virus Type 1 RNA Quantification and PCR Amplification for Drug Resistance Testing{triangledown}

Marjorie Monleau,1 Céline Montavon,1 Christian Laurent,1 Michel Segondy,2 Brigitte Montes,2 Eric Delaporte,1 François Boillot,3,4 and Martine Peeters1*

UMR 145, Institut de Recherche pour le Développement, and University of Montpellier 1, Montpellier, France,1 Laboratoire de Virologie/CHU de Montpellier, Montpellier, France,2 Alter-Santé Internationale & Développement, Montpellier, France,3 International Union against Tuberculosis and Lung Diseases, Paris, France4

Received 24 November 2008/ Returned for modification 9 January 2009/ Accepted 28 January 2009

The development and validation of dried sample spots as a method of specimen collection are urgently needed in developing countries for monitoring of human immunodeficiency virus (HIV) infection. Our aim was to test some crucial steps in the use of dried spots, i.e., viral recovery and storage over time. Moreover, we investigated whether dried plasma and blood spots (DPS and DBS, respectively) give comparable viral load (VL) results. Four manual RNA extraction methods from commercial HIV type 1 (HIV-1) VL assays—a QIAamp minikit (Qiagen), the Abbott Molecular sample preparation system, the Nuclisens assay (bioMarieux), and High Pure viral nucleic acid kit (Roche Applied Science)—were compared for VL quantification and PCR amplification for genotypic drug resistance testing on dried spots from spiked plasma and residual samples from HIV-1 patients (n = 47; median VL, 4.13 log10 copies/ml). RNA recovery from DPS was efficient using Nuclisens extraction (median difference, 0.03 log10 copies/ml) and slightly underestimated using the Abbott Molecular sample preparation system (median difference, 0.35 log10 copies/ml). PCR amplification results were in concordance. Measurements from DBS overestimated VL for plasma, with VL results showing <3.7 log10 copies/ml. VL was stable for up to 3 months in spiked DPS stored at 20°C but for only 1 month at 37°C. A faster decline was observed in PCR efficiency: DPS could be stored for 1 week at 37°C and for 1 month at 20°C. In conclusion, the RNA extraction method is an important factor in obtaining reliable RNA quantification and PCR amplification of HIV-1 on DPS/DBS. DBS could be used as an alternative for DPS depending on HIV RNA cutoffs for virological failure. VL measurements remain stable over a longer period than do PCR amplification results.

* Corresponding author. Mailing address: UMR 145, IRD, 911 Avenue Agropolis, BP 64501, 34394 Montpellier Cedex 5, France. Phone: 33-467416161. Fax: 33-467416146. E-mail: martine.peeters{at}mpl.ird.fr

{triangledown} Published ahead of print on 4 February 2009.

Journal of Clinical Microbiology, April 2009, p. 1107-1118, Vol. 47, No. 4
0095-1137/09/$08.00+0     doi:10.1128/JCM.02255-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Johannessen, A., Troseid, M., Calmy, A. (2009). Dried blood spots can expand access to virological monitoring of HIV treatment in resource-limited settings. J Antimicrob Chemother 64: 1126-1129 [Abstract] [Full Text]  


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