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

Abstract

BackgroundDried 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.MethodsA 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.ResultsUsing 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.ConclusionsWe 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.

Evaluation of Different RNA Extraction Methods and Storage Conditions of Dried Plasma or Blood Spots for Human Immunodeficiency Virus Type 1 RNA Quantification and PCR Amplification for Drug Resistance Testing

“…The facility to prepare, transport, and store dried spots is the major advantage of this sample support. Different studies have shown that dried spots can be kept for long periods when refrigerated or frozen in hermetic bags with desiccant, such as 1 year at 4°C (25) and 4 years at Ϫ20°C (17) for genotypic drug resistance testing and at least 15 days at 4°C (1, 11) and 1 year at Ϫ70°C (10) for RNA quantification. These data are important but are only applicable to reference laboratories where the infrastructure for long-term storage is avail- able.…”

Antiretroviral Drug Resistance Mutations Among Treated and Treatment-Naive Patients in Pakistan: Diversity of the HIV Type 1 pol Gene in Pakistan

“…The study was reviewed by the ethical review committee of Bridge Consultants Foundation and Vanderbilt University Institutional Review Board. DBS were prepared according to a protocol described by Youngpairoj et al 8 Fifty microliters of whole blood was spotted on 903 filter paper (Schleicher & Schuell, Keene, NH) and dried at room temperature for 3 h. The DBS were then sent to the China Center for Disease Control and Prevention for further genotypic testing at room temperature.…”

Field Study of Dried Blood Spot Specimens for HIV-1 Drug Resistance Genotyping

“…The sensitivities of PCR amplification from DBS differ significantly, with a few in-house assays reporting success from DBS specimens with VL as low as 1,000 to 2,000 copies/ml (14)(15)(16). All reports indicate lower genotyping sensitivities from DBS than from plasma specimens (17)(18)(19)(20)(21). Notably, genotyping sensitivity of 1,000 copies/ml was achieved by only two laboratories in a recent WHO proficiency testing program (22).…”