After excluding the subtype-related polymorphisms, the median number of PI-resistance mutations was 8 (range 0–12) (Table 1). The four PI-free patients and the patient receiving boosted atazanavir (ATVr) had fewer than eight PI-resistance mutations (no PI-resistance mutation in only one PI-naïve patient) and the remaining six patients had eight or more PI mutations and were currently receiving a PI-containing regimen (Table 1). Overall, seven patients exhibiting a protease insert-containing virus were followed up for a median duration of 24 months (range 10–62 months)

and this virus was detected for a median duration of 32 months (range 12–62 months) in six of them. Three patients were PI-naïve (patients 1, 2 and 3) when virus harbouring the protease insertion was first detected, VX-809 in vitro including one patient who never received any ARV therapy. All these patients were infected with an HIV-1 non-B subtype. No major PI-resistance mutations were detected in plasma virus harboured by these patients. In patient 1, the insertion E35E-T was present before ARV initiation. A nonnucleoside reverse transcriptase inhibitor (NNRTI)-containing regimen was initiated with a sustained virological response. Regarding the cell reservoir in this patient throughout

the 4 years of follow-up, the insert-containing virus was found to be archived C59 wnt in HIV DNA. Patient 2 exhibited Methane monooxygenase plasma virus with a 6-bp insertion (ins L38L-NL), first detected during pregnancy. The patient had a low plasma viral load (3.28 log10 HIV-1 RNA copies/mL) and was successfully treated with LPV (boosted with ritonavir) monotherapy to prevent materno-foetal transmission, reaching a viral load below the limit of detection of 50 copies/mL 1 month later.

Seventeen months after LPV discontinuation, the insert-containing virus was still detected as the major plasma viral population without additional nucleotide changes. Patient 3 was treated for 4 years with a stavudine/lamivudine/efavirenz regimen when the first genotype test was performed following loss of virological control; this showed an additional asparagine amino acid following the S37N mutation (ins S37N-N). In our study, eight of the 11 patients harbouring protease insert-containing virus were PI-experienced; of these patients, six were infected with HIV-1 subtype B. One of the patients (patient 4) had been off ARVs for 5 years when a first genotype test detected the insertion; of note, he previously received 9 months of NFV and IDV treatment. Two months following the initiation of a new PI-containing regimen (ATV), the HIV-1 RNA plasma viral load decreased to 3.56 log10 copies/mL.