Intensification therapy adding a boosted protease inhibitor (PI) to a failing regimen has the potential to worsen the resistance profile. Sixty-six patients included in four different boosted PI intensification studies were assessed, and the resistance mutations in the reverse transcriptase (RT) and protease genes were evaluated at baseline and 4 weeks after the initiation of the intensification strategy. Only one of the 66 patients (1.5%) developed changes in their pattern of mutations able to generate or increase resistance to new drugs.
Intensification therapy is a strategy that can be used to improve the virological response in patients with an intermediate level of resistance  or a low degree of virological failure [2–6]. There are two ways of intensifying therapy: by incorporating one or more new drugs in a failing regimen  or by boosting with a low dose of ritonavir a regimen containing a PI-unboosted PI .
The selection of resistance mutations is one of the major causes of treatment failure . The development of new HIV RT or protease resistance mutations has been demonstrated within the short period of time between the blood drawn for resistance testing and the availability of the results , or shortly after the initiation of an intensification strategy . Because there are almost no data about the evolution of resistance-associated mutations early after the initiation of a strategy of intensifying therapy, we examined this issue by pooling together data from several pilot studies of boosted PI intensification. We selected patients with the following criteria: (i) patients failing an unboosted PI or a non-nucleoside reverse transcriptase inhibitor-containing regimen in which the non-nucleoside reverse transcriptase inhibitor or the PI was replaced by the same or another PI (saquinavir , indinavir , amprenavir ) but boosted with a low dose of ritonavir or lopinavir/ritonavir  until the results of the resistance test became available; and (ii) patients with a plasma viral load (PVL) remaining above 1000 copies/ml after 4 weeks of treatment.
Seventy-six patients were eligible. Eighteen remained with a PVL above 1000 copies/ml at week 4. Of these, HIV RNA could be amplified in 10, who were included in this study. The eight patients that could not be amplified had a PVL ranging between 1000 and 3000 copies/ml. Adherence in all of these patients was above 95%, measured by pill count and by a self-administered questionnaire.
Genotypic resistance analysis was performed using the TruGene HIV-1 assay (Visible Genetics Inc., Toronto, Canada) and the interpretation of resistance sequences was done by the Sequence Stanford Database. RT and protease sequences were interpreted using a publicly available algorithm, HIV RT and Protease Sequence Database (http://hivdb.stanford.edu), updated in August 2002. Plasma drug levels of saquinavir, indinavir, amprenavir, lopinavir and ritonavir were determined at week 4 using a rapid, simple and sensitive high-performance liquid chromatography assay, developed and validated for the simultaneous quantification of the HIV PI indinavir, ritonavir, amprenavir, saquinavir, nelfinavir and lopinavir in human plasma, at the Pharmacology Department of the Hospital Clinic (Barcelona, Spain) .
Changes in genotypic mutation patterns from baseline to week 4 after introducing the intensification therapy could be assessed in 10 patients. The median baseline values of viral load and CD4 cell count were 26 450 copies/ml (range 11 100–1 000 000) and 316 cells/μl (range 31–594), respectively. The median decrease in viral load and increase in CD4 cell count at week 4 were 0.605 log10/ml (from 0.11 to 1.98) and over 25 cells/mm3, respectively.
The evolution of HIV resistance-associated mutations is shown in Table 1. Five of these 10 patients (50%; representing 6% of the total number of 76) either lost or gained a RT or a protease resistance-associated mutation. All mutations detected at baseline were also present at week 4, with the exception of an L210W mutation in one patient. Five patients accumulated new mutations by week 4 (two out of 10 patients accumulated primary mutations, one out of 10 secondary mutations, and four out of 10 polymorphic site mutations). However, in only one patient did the new mutation have potential clinical significance according to Stanford Database interpretation. This patient, who had a previous history of zalcitabine drug exposure, and was receiving lamivudine plus stavudine plus indinavir and was intensified with ritonavir-boosted indinavir, gained a T69N mutation that meant a change of susceptibility to stavudine and zalcitabine from susceptible and intermediate resistant to intermediate resistant and resistant, respectively. Four out of 10 patients developed nucleoside reverse transcriptase inhibitor mutations (K70R, T69N, K219Q and a polymorphism G333E). The acquisition of mutations K70R and K219Q in patient number 3, who was failing a zidovudine-containing regimen, was associated with the loss of the L210W mutation, with no change in the pattern of drug susceptibility according to the Stanford Database algorithm interpretation. Two out of 10 patients (20%) developed new PI mutations (L63P and I93L), which do not cause any appreciable change in the susceptibility to any PI. In a logistic regression model, statistically significant differences were found between the five patients who showed changes in genotypic mutation patterns during intensification therapy in the magnitude of viral load drop (Δ PVL at week 4 > 0.5 log10) at week 4 (P < 0.05) compared with the five who did not develop changes. Conversely, no statistical differences were found in terms of the number of previous treatments, baseline PVL, CD4 cell count, or virtual inhibitory quotient for the boosted PI used in intensification therapy.
In summary, intensifying with a boosted PI in virologically failing patients does not seem to increase the number of clinically significant mutations or worsen the resistance profile at one month.
Sponsorship: This study was partly supported by Red de Grupos de Investigacion en SIDA (RIS) del FISS.
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