HIV-1 drug resistance genotyping from dried blood spots stored for 1 year at 4 degrees C.
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.
Youngpairoj
Ae S
AS
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.
Masciotra
Silvina
S
Garrido
Carolina
C
Zahonero
Natalia
N
de Mendoza
Carmen
C
García-Lerma
J Gerardo
JG
D001769:Blood / Q000821:virology*; D003890:Desiccation; D004351:Drug Resistance / Q000235:genetics*; D005838:Genotype; D015658:HIV Infections / Q000821:virology*; D015497:HIV-1 / Q000187:drug effects / Q000235:genetics* / Q000302:isolation & purification*; D006801:Humans; D008826:Microbial Sensitivity Tests / Q000379:methods*; D009696:Nucleic Acids / Q000302:isolation & purification; D012034:Refrigeration; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D013048:Specimen Handling / Q000379:methods*; D013997:Time Factors
D009696:Nucleic Acids