Loss of genotypic activity of NRTIs
The loss of genotypic activity of NRTIs was considerably higher in patients treated with NNRTIs compared to PI/r. The loss of genotypic activity of NRTIs was already very high ,3 months after failure when patients had been treated with NNRTIs (38.2%) whereas PI/r-treated patients rarely accumulated NRTI mutations in this time period (8.8%, p = 0.009). The loss of genotypic activity of NRTIs remained considerably higher among patients treated with NNRTIs compared to PI/r also after long-lasting exposure to the failing regimen: 46.9% vs. 7.1% (p,0.001) between 3? months and 60.9% vs. 18.9% (p,0.001) after .6 months with replicating virus. The loss of activity of more than one NRTI was quite rare in the PI/r group for all time points: 2.9%, 7.1%, 0% when compared to the NNRTI group: 14.7% (p = 0.197), 14.3% (p = 0.474), 21.7% (p = 0.002) at ,3, 3? and .6 months after failure (Figure 1 A). We excluded non-responders (n = 48) from a sensitivity analysis. The results remained more favorable for the PI/r group: 37.5% vs. 9.7% (p = 0.016), 38.9% vs. 9.1% (p = 0.016), 57.1% vs. 16.7% (p = 0.003) NNRTI- and PI-treated individuals lost the activity of $1 NRTI after ,3, 3?, .6 months with replicating virus. Results were confirmed if the analysis was restricted to the 113 patients who truly failed first-line cART (patients who changed a successful cART regimen due to toxicity reasons were excluded) although the sample size was reduced: 23.1% vs. 38.5% (p = 0.673), 8.3% vs. 48.0% (p = 0.027), 22.2% vs. 65.6% (p = 0.007) after ,3, 3?, .6 months with replicating virus lost the activity of $1 NRTI in the PI/r and NNRTI group, respectively. The treatment with more potent NRTI combinations could be a potential explanation for the lower accumulation of NRTI mutations. PI/r-treated individuals received more often the modern and well tolerated NRTI combination TDF/FTC (40.4%) compared to NNRTI-treated individuals (17.1%). In a sensitivity
Figure 1. Loss of genotypic activity. Loss of genotypic activity among patients treated with non-nucleoside reverse transcriptase inhibitors (NNRTIs) or ritonavir-boosted protease inhibitors (PI/r). A) Loss of activity of 1 NRTI (open bar section) or .1 NRTIs (hatched bar section). B) Loss of activity of a NNRTI or PI/r. 95% confidence intervals are indicated. *** Fisher exact p,0.001, ** p,0.01. analysis, we only included patients who were treated with the TDF/FTC NRTI combination (n = 62). Also in this sub-group, the loss of activity of $1 NRTI was higher in NNRTI compared to PI/r-treated individuals, 26.1% compared to 5% (p = 0.043), respectively. Higher copy-years viremia among patients treated with NNRTI would be a potential explanation for faster accumulation of mutations. We repeated the analysis and classified patients in 3 groups based on the copy-years viremia instead of the time with replicating virus. Each group contained 76 patients with copyyears viremia ,115, 115?491,.1491 years * copies/mL. The loss of genotypic activity of $1 NRTIs was 60.6% vs. 7.0% (p,0.001), 51.2% vs. 15.2% (p = 0.002) and 41.5% vs. 17.4% (p = 0.064) in group 1 to 3 among NNRTI and PI/r treated individuals, respectively. These findings demonstrate that the higher loss of activity in NNRTI-treated patients cannot solely be explained by a more intense viral replication.
Risk factors for the early emergence of NRTI mutations
Exposure to NNRTIs was the major risk factor for an early accumulation of NRTI mutations. The odds ratio (OR) was 10.6 (95% CI: 3.9?9.1) and 13.3 (95% CI: 4.1?2.8) in univariable and multivariable models, respectively. High CD4 cell count at the time of the resistance test was the only other factor that had a weak association with a later accumulation of NRTI mutations (univariable OR: 0.9 (95% CI: 0.9?.0), multivariable OR: 0.9 (95% CI: 0.8?.0). No other factor was significantly associated in the multivariable model (Table 2).
Discussion
We showed that cART containing PI/r results in a long-lasting protection of the activity of NRTIs and PI/r during sustained viral Loss of genotypic activity of PIs and NNRTIs
The loss of genotypic activity of NNRTIs occurred very early after treatment failure whereas the emergence of PI/r resistance was very rare even after long-lasting exposure to failing cART. The percentage of patients who have lost PI/r activity was 2.9%, 3.6%, 5.4% ,3, 3? and .6 months compared to 41.2%, 49.0%, 63.0% of those who have lost NNRTI activity (all p,0.001) (Figure 1 B). Results were similar when individuals were categorized according to copy-years viremia strata: 7.0% vs. 57.6%, 0% vs. 44.2%, 4.4% vs. 54.7% (all p,0.001). Excluding non-responders or studying exclusively patients who failed first-line treatment confirmed these results (data not shown).
Emerging mutations
The most common cause for the loss of genotypic activity of NRTIs was the emergence of M184V. It occurred in 36.4% of NNRTI- and in only 9.1% of PI/r-treated individuals (p,0.001). The prevalence of additional NRTI mutations was also much higher in the NNRTI when compared to the PI/r group: K65R 10.9% vs. 1.0% (p = 0.003), M184I 7.0% vs. 1.0% (p = 0.029), T215Y 5.4% vs. 1.0% (p = 0.072). All other mutations had a prevalence of ,5% in both groups (Figure 2). The loss of NNRTI activity was mainly caused by the emergence of K103N (27.9%), Y181C (10.9%) or G190A (6.2%). The most common PI mutations in PI/r-treated individuals were L90M (3.0%), I84V (2.0%) and M46I (2.0%).
Figure 2. Emergence of mutations. Accumulation of nucleoside reverse transcriptase inhibitor (NRTI) mutations after virological failure on combination antiretroviral therapy containing A) non-nucleoside reverse transcriptase inhibitors (NNRTIs) or B) ritonavir-boosted protease inhibitors (PI/r): K65R, M184I/V, thymidine analogue mutations (TAM) 1 or 2, and other NRTI mutations defined by IAS-USA [15]. 95% confidence intervals are indicated.Table 2. Univariable and multivariable logistic regression analyzing risk factors for an early (,6 months with replicating viruses) emergence of $1 nucleoside reverse transcriptase (NRTI) mutations (n =145).