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Mutation proI47A has recently been associated with lopinavir/ritonavir (LPV/r) resistance. Only four out of 1859 specimens (0.2%) sent for drug resistance testing (219 drug-naive and 1650 antiretroviral-experienced) showed I47A. All belonged to patients failing LPV/r. The prevalence among protease inhibitor-experienced patients was 0.6%. Phenotypic testing showed that proI47A caused high-level lopinavir resistance (> 100-fold) and cross-resistance to amprenavir, whereas it caused hypersusceptibility to saquinavir. ProI47A should thus be considered the primary lopinavir resistance mutation.