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

Abstract

The development and validation of dried sample spots as a method of specimen collection are urgently needed in developing countries for monitoring of human immunodeficiency virus (HIV) infection. Our aim was to test some crucial steps in the use of dried spots, i.e., viral recovery and storage over time. Moreover, we investigated whether dried plasma and blood spots (DPS and DBS, respectively) give comparable viral load (VL) results. Four manual RNA extraction methods from commercial HIV type 1 (HIV-1) VL assays-a QIAamp minikit (Qiagen), the Abbott Molecular sample preparation system, the Nuclisens assay (bioMarieux), and High Pure viral nucleic acid kit (Roche Applied Science)-were compared for VL quantification and PCR amplification for genotypic drug resistance testing on dried spots from spiked plasma and residual samples from HIV-1 patients (n ‫؍‬ 47; median VL, 4.13 log 10 copies/ml). RNA recovery from DPS was efficient using Nuclisens extraction (median difference, 0.03 log 10 copies/ml) and slightly underestimated using the Abbott Molecular sample preparation system (median difference, 0.35 log 10 copies/ml). PCR amplification results were in concordance. Measurements from DBS overestimated VL for plasma, with VL results showing <3.7 log 10 copies/ml. VL was stable for up to 3 months in spiked DPS stored at 20°C but for only 1 month at 37°C. A faster decline was observed in PCR efficiency: DPS could be stored for 1 week at 37°C and for 1 month at 20°C. In conclusion, the RNA extraction method is an important factor in obtaining reliable RNA quantification and PCR amplification of HIV-1 on DPS/DBS. DBS could be used as an alternative for DPS depending on HIV RNA cutoffs for virological failure. VL measurements remain stable over a longer period than do PCR amplification results.Due to the efforts of national programs and the support of a wide range of international partners, the number of people receiving antiretroviral therapy (ART) in resource-limited countries has increased significantly over recent years, most notably in sub-Saharan Africa (24). In order to limit the emergence of resistance to antiretroviral drugs, human immunodeficiency virus (HIV) treatment should ideally be accompanied by periodic virological monitoring, such as viral load and resistance testing. However, in resource-limited countries, viral load monitoring and drug resistance tests are not yet available at an affordable cost, and infrastructure requirements limit the scale-up in access to these tests. Treatment initiation and modifications are thus guided mainly by clinical disease progression and by CD4 cell counts, when possible (13, 24). As a consequence, many people with virological failure stay on inadequate drug regimens for long periods. Rapid or uncontrolled emergence of HIV drug resistance is thus feared as a potential consequence of ART scale-up in resource-limited countries. Therefore, ART resistance monitoring is not recommended at the individual patient level in low-income countries, and the WHO has established a sys...

Design and Implementation of an External Quality Assessment Program for HIV Viral Load Measurements Using Dried Blood Spots

“…These results are consistent with other reports and likely reflect the amplification of cell-associated HIV DNA and RNA in DBS samples in the absence of detectable blood plasma RNA (7,9,10). The magnitude of this discrepancy may be reduced using DBS preparation procedures that preferentially select for cell-free HIV RNA (11); however, the amplification of residual DNA in DBS to even a minimal extent may confound the interpretation of HIV RNA copy number in subjects with ART-induced virologic suppression.…”

Higher Specificity of Nucleic Acid Sequence-Based Amplification Isothermal Technology than of Real-Time PCR for Quantification of HIV-1 RNA on Dried Blood Spots

“…Overall, VL values obtained with DBS specimens tended to be lower than those obtained with the corresponding plasma samples, with a mean difference of around 0.4 log 10 copies/ml. This difference has previously been noted with the NucliSENS, Abbott, and Roche methods (10,11) and is likely due to differences in specimen volume and in the quality of nucleic acid extraction. VL values obtained with DBS specimens should thus not be compared with VL values previously obtained for plasma samples when monitoring therapeutic efficacy in a given patient.…”

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

“…Studies have shown that when DBS were stored at higher temperature and humidity, the proportion successfully amplified and genotyped was reduced (19,21,27,29,30). Despite all these studies, there are limited data on the effect of shipment conditions on genotyping efficiency or whether previously frozen DBS can be shipped at ambient temperature using routine courier services without significantly compromising genotyping efficiency.…”