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

• Published in: Journal of Clinical Microbiology Vol. 47; no. 4; pp. 1107 - 1118
• Main Authors: Monleau, Marjorie, Montavon, Céline, Laurent, Christian, Segondy, Michel, Montes, Brigitte, Delaporte, Eric, Boillot, François, Peeters, Martine
• Format: Journal Article
• Language: English
• Published: Washington, DC American Society for Microbiology 01.04.2009; American Society for Microbiology (ASM)
• Subjects: Biological and medical sciences; Blood - virology; Desiccation; Drug Resistance, Viral; Fundamental and applied biological sciences. Psychology; HIV Infections - virology; HIV-1 - genetics; HIV-1 - isolation & purification; Human immunodeficiency virus 1; Human viral diseases; Humans; Infectious diseases; Medical sciences; Microbiology; Miscellaneous; Molecular Diagnostic Techniques; Plasma - virology; Polymerase Chain Reaction - methods; Reagent Kits, Diagnostic; RNA, Viral - genetics; RNA, Viral - isolation & purification; Specimen Handling - methods; Temperature; Time Factors; Viral diseases; Viral diseases of the lymphoid tissue and the blood. Aids; Viral Load; Virology; Immunopathology; Microbiology; HIV-1 virus; Retroviridae; AIDS; Method; Immune deficiency; Lentivirus; Blood plasma; Infection; Virus; Amplification; Polymerase chain reaction; Resistance; Storage; Viral disease; Human immunodeficiency virus; Quantitative analysis
• ISSN: 0095-1137; 1098-660X
• DOI: 10.1128/JCM.02255-08

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 log₁₀ copies/ml). RNA recovery from DPS was efficient using Nuclisens extraction (median difference, 0.03 log₁₀ copies/ml) and slightly underestimated using the Abbott Molecular sample preparation system (median difference, 0.35 log₁₀ copies/ml). PCR amplification results were in concordance. Measurements from DBS overestimated VL for plasma, with VL results showing <3.7 log₁₀ 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.