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