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To understand the pathogenesis of low level viraemia (LLV) in HIV-infected patients on boosted protease inhibitors (PI/b), we enrolled 34 subjects with a median HIV-RNA 79 copies/mL and followed them for 15 months. Samples for next generation sequencing were collected at three time-points. Two showed resistance-associated mutations (RAMs) in the protease gene, while 95–100% had RAMs in the gag gene, which evolved in approximately a quarter of subjects, suggesting a potential clinical role of these kind of mutations.