title: Rapid decline in the efficiency of HIV drug resistance genotyping from dried blood spots (DBS) and dried plasma spots (DPS) stored at 37 degrees C and high humidity.
abstract: OBJECTIVE
  Dried blood spots (DBS) and dried plasma spots (DPS) are considered convenient alternatives to serum and plasma for HIV drug resistance testing in resource-limited settings. We sought to investigate how extreme conditions could affect the short-term ability to amplify and genotype HIV from DBS.
  
  
  METHODS
  A panel of six matched DPS/DBS was generated using blood collected from HIV-infected donors. Replicate cards were prepared in 903 filter paper using 50 microL of blood and stored at either -20 degrees C or at 37 degrees C/100% humidity. Nucleic acids were extracted at baseline and after 1, 2, 8 and 16 weeks of storage and were amplified and sequenced using an in-house RT-nested PCR method or the ViroSeq assay.
  
  
  RESULTS
  HIV-1 pol was successfully amplified in all DBS/DPS at baseline and in those stored for up to 16 weeks at -20 degrees C by the in-house assay. In contrast, amplification was rapidly lost during storage at 37 degrees C/100% humidity with only 6/6 and 4/6 DBS specimens amplifiable by the in-house assay at weeks 1 and 2, respectively. Similarly, only two DPS stored at 37 degrees C/100% humidity were amplified by the in-house assay at week 1.
  
  
  CONCLUSIONS
  We show that resistance testing from DBS and DPS is severely compromised after 2 and 1 weeks of storage at 37 degrees C/100% humidity with desiccant, respectively. These findings underscore the importance of temperature and humidity for the efficient genotyping of HIV-1 from DBS and DPS, and reiterate the need to rapidly transport specimens from collection sites to locations that have appropriate storage conditions such as -20 degrees C.
authors.affiliation: 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 30333, USA.
authors.forename: J Gerardo
authors.initials: JG
authors.lastname: García-Lerma
authors.forename: Amanda
authors.initials: A
authors.lastname: McNulty
authors.forename: Cheryl
authors.initials: C
authors.lastname: Jennings
authors.forename: Diana
authors.initials: D
authors.lastname: Huang
authors.forename: Walid
authors.initials: W
authors.lastname: Heneine
authors.forename: James W
authors.initials: JW
authors.lastname: Bremer
mesh_terms: D001769:Blood / Q000821:virology*; D003890:Desiccation; D024882:Drug Resistance, Viral; D005838:Genotype; D015497:HIV-1 / Q000235:genetics* / Q000302:isolation & purification; D006801:Humans; D006813:Humidity; D010949:Plasma / Q000821:virology*; D016133:Polymerase Chain Reaction / Q000379:methods; D012367:RNA, Viral / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D013048:Specimen Handling / Q000379:methods*; D013696:Temperature; D054302:pol Gene Products, Human Immunodeficiency Virus / Q000235:genetics
chemical_list: D012367:RNA, Viral; D054302:pol Gene Products, Human Immunodeficiency Virus