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.
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.
García-Lerma
J Gerardo
JG
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.
McNulty
Amanda
A
Jennings
Cheryl
C
Huang
Diana
D
Heneine
Walid
W
Bremer
James W
JW
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
D012367:RNA, Viral; D054302:pol Gene Products, Human Immunodeficiency Virus