HIV-1 resistance genotyping on dried serum spots.
OBJECTIVE
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.


METHODS
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.


RESULTS
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).


CONCLUSIONS
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.
Plantier
Jean-Christophe
JC
Unité de Virologie, laboratoire associé au Centre National de Référence du VIH, Equipe d'Accueil 2656, CHU Charles Nicolle, Rouen, France. Jean-Christophe.Plantier@univ-rouen.fr
Dachraoui
Raoudha
R
Lemée
Véronique
V
Gueudin
Marie
M
Borsa-Lebas
Françoise
F
Caron
François
F
Simon
François
F
D023241:Antiretroviral Therapy, Highly Active; D001800:Blood Specimen Collection / Q000379:methods; D016903:Drug Monitoring / Q000379:methods*; D024882:Drug Resistance, Viral / Q000235:genetics*; D005240:Feasibility Studies; D005838:Genotype; D015658:HIV Infections / Q000188:drug therapy* / Q000821:virology; D015497:HIV-1 / Q000187:drug effects* / Q000235:genetics*; D006801:Humans; D010802:Phylogeny; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D016415:Sequence Alignment; D019562:Viral Load