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

J Antimicrob Chemother. 2008 Jun;61(6):1217-20. doi: 10.1093/jac/dkn100. Epub 2008 Mar 15.

Abstract

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 degrees C for prolonged periods (0.5-4 years). Here, we evaluated the efficiency of genotyping from DBSs stored at 4 degrees 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 degrees 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 degrees 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 degrees C may represent a suitable temperature for long-term storage of DBSs.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Blood / virology*
  • Desiccation
  • Drug Resistance / genetics*
  • Genotype
  • HIV Infections / virology*
  • HIV-1 / drug effects
  • HIV-1 / genetics*
  • HIV-1 / isolation & purification*
  • Humans
  • Microbial Sensitivity Tests / methods*
  • Nucleic Acids / isolation & purification
  • Refrigeration
  • Reverse Transcriptase Polymerase Chain Reaction
  • Sensitivity and Specificity
  • Specimen Handling / methods*
  • Time Factors

Substances

  • Nucleic Acids