HIV-1 resistance genotyping on dried serum spots

• Published in: AIDS (London) Vol. 19; no. 4; pp. 391 - 397
• Main Authors: PLANTIER, Jean-Christophe, DACHRAOUI, Raoudha, LEMEE, Véronique, GUEUDIN, Marie, BORSA-LEBAS, Francoise, CARON, Francois, SIMON, Francois
• Format: Journal Article
• Language: English
• Published: Hagerstown, MD Lippincott Williams & Wilkins 04-03-2005
• Subjects: AIDS/HIV; Antibiotics. Antiinfectious agents. Antiparasitic agents; Antiretroviral Therapy, Highly Active; Antiviral agents; Biological and medical sciences; Blood Specimen Collection - methods; Drug Monitoring - methods; Drug Resistance, Viral - genetics; Feasibility Studies; Genotype; HIV Infections - drug therapy; HIV Infections - virology; HIV-1 - drug effects; HIV-1 - genetics; Human immunodeficiency virus 1; Human viral diseases; Humans; Infectious diseases; Medical sciences; Pharmacology. Drug treatments; Phylogeny; Reverse Transcriptase Polymerase Chain Reaction - methods; Sequence Alignment; Viral diseases; Viral diseases of the lymphoid tissue and the blood. Aids; Viral Load; HIV-1; Antiretroviral agent; HIV-1 virus; Microbiological investigation; Molecular epidemiology; Antiviral; Serum; dried serum spot; Reverse transcription polymerase chain reaction; Sequencing; Human; Immunopathology; Typing; Nucleotide sequence; Phylogenetic tree; Retroviridae; Genotype; AIDS; Genetic diversity; Filter paper; Method; resistance genotyping; Immune deficiency; Lentivirus; Infection; Virus; Resistance; Surveillance; Viral disease; Feasibility; Human immunodeficiency virus; Mutation; Molecular biology; Subtype; Polymorphism
• ISSN: 0269-9370; 1473-5571
• DOI: 10.1097/01.aids.0000161768.98534.e7

To assess the feasibility of HIV-1 group M resistance genotyping on dried serum spots, by testing samples from previously untreated patients, patients on treatment, and patients having stopped treatment, representing a wide genetic diversity panel.
Serum samples from 62 HIV-1-infected Caucasian and African patients, with viral load values from 715 copies/ml to more than 750,000 copies/ml, were deposited on filter paper. After elution and RNA extraction, nested RT-PCR was used to amplify the protease and RT regions of the pol gene. Resistance sequencing was performed on all the protease and RT amplicons. The sequences obtained for resistance genotyping were used for subtyping by phylogenetic analysis.
Amplification was successful in the protease region in 53/62 cases (85.5%) and in the RT region in 51/62 cases (82.3%). All samples with viral loads of at least 5 Log (17 of 62) were successfully amplified in both the RT and protease regions. Of the 29 samples with viral loads between 4 Log and 5 Log, 28 (97%) were amplified in the RT region and 25 (86%) in the protease region. The detected mutations were in keeping with the treatment status. Marked natural polymorphism was observed in the protease region, but no major consequences were deduced in terms of resistance. The results showed a broad diversity of the panel, including subtype B (n = 36) and non B or recombinant forms (n = 20).
Our results show the feasibility of this dried serum spot method for monitoring resistance to antiretroviral drugs and the molecular epidemiology of HIV diversity. The simplicity of sample preparation, storage and transport potentially makes this an importance tool for individual and epidemiological monitoring throughout the world.