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 degrees C but for only 1 month at 37 degrees C. A faster decline was observed in PCR efficiency: DPS could be stored for 1 week at 37 degrees C and for 1 month at 20 degrees 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.

FIG. 1.

HIV-1 viral loads in patient samples (n = 47) of plasma and DPS after RNA extractions with a QIAamp viral RNA mini kit (Qiagen), the Abbott sample preparation system, or a Nuclisens manual extraction kit (bioMérieux). (A) Comparison of median viral loads from DPS with those in matched plasma samples. The gray squares represent medians, the boxes represent interquartile ranges, the whiskers represent lower and upper adjacent values, and the black dots represent outside values. (B) Comparison of viral loads in DPS and corresponding plasmas, only for samples with detectable viral loads, after RNA extraction with Qiagen (gray rhombuses), Abbott (black squares), and bioMérieux (white triangles) extraction kits. Viral loads are expressed as log₁₀ copies/ml. P values were calculated by the Wilcoxon test by comparing liquid plasma and spots.

FIG. 2.

Bland-Altman analysis of HIV-1 viral loads in patient samples (n = 47) of plasma versus DPS after RNA extraction with a QIAamp viral RNA mini kit (Qiagen) (A), the Abbott sample preparation system (B), and a Nuclisens manual extraction kit (bioMérieux) (C). Horizontal black lines represent the mean difference, and dotted lines show the standard deviation.

FIG. 3.

HIV-1 viral loads in patient samples (n = 39) of plasma versus DPS (black) and DBS (gray). HIV-1 RNA was measured in DPS and DBS with detectable viral loads after RNA extraction with the Abbott sample preparation system (A) and a Nuclisens manual extraction kit from bioMérieux (B) and then related to viral loads in plasma. Black vertical lines indicate 3.70 log₁₀ copies/ml in liquid plasma. Bland-Altman analysis was performed for spots after RNA extraction with the RNA extraction kits of Abbott (C) and bioMérieux (D). Horizontal lines represent the mean difference, and dotted lines show the standard deviation (in black for DPS and in gray for DBS).

FIG. 4.

HIV-1 viral loads (log₁₀ copies/ml) in DPS and DBS after RNA extraction with the Abbott sample preparation system or a Nuclisens manual extraction kit from bioMérieux for patient samples stratified according to plasma viral load. (A) Viral loads in plasma of <3.70 log₁₀ copies/ml. (B) Viral loads in plasma of >3.70 log₁₀ copies/ml. P values are calculated by the Wilcoxon test by comparing liquid plasma and spots. The gray squares represent medians, the boxes represent interquartile ranges, the whiskers represent lower and upper adjacent values, and the black dots represent outside values.

FIG. 5.

HIV-1 viral loads over time and under different storage conditions, evaluated from DPS (duplicate mean) prepared from spiked plasma samples with 3.30 (red), 4.31 (blue), and 5.32 (gray) log₁₀ copies/ml and after RNA extraction with the Abbott sample preparation system (squares) and a Nuclisens manual extraction kit (bioMérieux) (triangles). (A) Storage conditions of 20°C and a dry atmosphere. (B) Storage conditions of 37°C and a humid atmosphere.