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JAC Advance Access originally published online on March 15, 2008
Journal of Antimicrobial Chemotherapy 2008 61(6):1217-1220; doi:10.1093/jac/dkn100
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Published by Oxford University Press 2008
The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and Oxford University Press are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated. For commercial re-use, please contact journals.permissions@oxfordjournals.org

Original research

HIV-1 drug resistance genotyping from dried blood spots stored for 1 year at 4°C

Ae S. Youngpairoj1, Silvina Masciotra1, Carolina Garrido2, Natalia Zahonero2, Carmen de Mendoza2 and J. Gerardo García-Lerma1,*

1 Laboratory Branch, Division of HIV/AIDS Prevention, National Center for HIV/AIDS, Viral Hepatitis, STD and TB Prevention, Centers for Disease Control and Prevention, Atlanta, GA, USA 2 Department of Infectious Diseases, Hospital Carlos III, Madrid, Spain

Received 4 December 2007; returned 20 January 2008; revised 6 February 2008; accepted 17 February 2008

* Corresponding author. Tel: +1-404-639-4987; Fax: +1-404-639-1174; E-mail: ggarcia-lerma{at}cdc.gov

Background: Dried blood spots (DBSs) are an attractive alternative to plasma for HIV-1 drug resistance testing in resource-limited settings. We recently showed that HIV-1 can be efficiently genotyped from DBSs stored at –20°C for prolonged periods (0.5–4 years). Here, we evaluated the efficiency of genotyping from DBSs stored at 4°C for 1 year.

Methods: A total of 40 DBSs were prepared from residual diagnostic specimens collected from HIV subtype B-infected persons and were stored with desiccant at 4°C. Total nucleic acids were extracted after 1 year using a modification of the Nuclisens assay. Resistance testing was performed using the ViroSeq HIV-1 assay and an in-house nested RT–PCR method validated for HIV-1 subtype B that amplifies a smaller (1 kb) pol fragment.

Results: Using the ViroSeq assay, only 23 of the 40 (57.5%) DBS specimens were successfully genotyped; 22 of these specimens had plasma viraemia >10 000 RNA copies/mL. When the specimens were tested using the in-house assay, 38 of the 40 DBSs (95%) were successfully genotyped. Overall, resistance genotypes generated from the DBSs and plasma were highly concordant.

Conclusions: We show that drug resistance genotyping from DBSs stored at 4°C with desiccant is highly efficient but requires the amplification of small pol fragments and the use of an in-house nested PCR protocol with quality-controlled reagents. These findings suggest that 4°C may represent a suitable temperature for long-term storage of DBSs.

Keywords: resistance testing , ViroSeq assay , 903 filter paper


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