No Evidence for Evolution of Protease Inhibitor Resistance from Standard Genotyping,After Three Years of Treatment with Darunavir/Ritonavir,With or Without Nucleoside Analogues

Abstract

E ditor: In several recent studies, the risk of treatment-emergent drug resistance has been lower for patients using combinations of two nucleoside analogues plus a boosted protease inhibitor (PI), compared to two nucleoside analogues plus a nonnucleoside.^1
–3 However, there are concerns that if patients with full HIV RNA suppression are switched to protease inhibitor monotherapy, there could be an increased risk of elevations in HIV RNA and subsequent resistance to PIs.^4,5

In the MONET trial^6,7 256 patients with HIV RNA <50 copies/ml and no history of virological failure were randomized to darunavir/ritonavir (DRV/r), either as monotherapy or with two nucleoside analogues, for 144 weeks. The primary endpoint of the trial was a confirmed elevation in HIV RNA above 50 copies/ml on two consecutive visits. All samples with HIV RNA >50 copies/ml in the MONET trial were genotyped. In addition, Virtual Phenotype was generated for all samples using the VircoType method. The number and type of IAS-USA minor and major PI mutations were investigated and compared over time in patients with two or more successive genotypes during episodes of viremia.

Genotypic mutations⁸ were defined as follows: darunavir mutations: V11I, V32I, L33F, I47V, I50V, I54L/M, T74P, L76V, I84V, L89V; IAS-USA major PI mutations: D30N, V32I, L33F, M46I/L, I47A/V, I50L, I54M/L, L76V, V82A/F/T, I84V, N88S, L90M; IAS-USA minor PI mutations: L10I/F/V/C, I13V, G16E, K20R/M/I/T/V, L24I, L33I/V, E34Q, M36I/L/V, G48V, F53L/Y, Q58E, D60E, I62V, L63P, I64I/M/V, A71V/I/T/L, G73C/S/T/A, V77I, N83D, I85V, I93L/M; nucleoside reverse transcriptase inhibitor (NRTI) mutations: M41L, A62V, K65R, D67N, K70R, L74V, V75I, F77L, Y115F, F116Y, Q151M, M184V, L210W, T215Y/F, K219Q/E.

The percentage of patients with each mutation was calculated from the number of patients with at least one successful genotype. The analysis assumed a worst case scenario: if any of a patient samples showed a mutation, the patient was assumed to have this mutation, even if other samples showed wild-type virus.

There were 111 samples from 54 patients with HIV RNA >50 copies/ml (31 on monotherapy and 23 on triple therapy) successfully genotyped. The HIV RNA level was below 400 copies/ml in 73% of these samples. The success rate from genotyping was 62%; 45/54 patients (83%) had subtype B HIV.

Of the 54 patients, 52 (96%) showed no major IAS-USA PI or NRTI mutations, or darunavir mutations. One patient in the DRV/r monotherapy arm showed a DRV mutation (L33F) during transient viremia before week 24. This patient sample remained phenotypically sensitive to darunavir (by VircoType) and the patient had HIV RNA suppression below 50 copies/ml to the end of the trial, with no changes in treatment.

One patient in the DRV/r + 2NRTIs arm, a protocol violator, had the M184V mutation and two PI mutations (V82I, L90M) in a prescreening genotype. This patient had reemergence of the M184V mutation at week 12 of the trial, together with three PI mutations (I54V, V82T, and L90M). This patient sample showed phenotypic resistance to atazanavir, fosamprenavir, indinavir, lopinavir, saquinavir, and tipranavir. However, the sample remained phenotypically sensitive to darunavir (by VircoType) and the patient had long-term suppression of HIV RNA to the end of the trial, with no changes in treatment.

Analysis by VircoType also showed no phenotypic resistance to atazanavir, darunavir, fosamprenavir, indinavir, lopinavir, saquinavir, or tipranavir in 52/54 patients (96%). The one patient in the DRV/r + 2NRTIs arm with phenotypic resistance to PIs is described above. There was one patient in the DRV/r arm with a sample at week 128 showing reduced phenotypic sensitivity to indinavir, when the HIV RNA had a single elevation of 264 copies/ml. This patient sample was phenotypically sensitive to all other boosted PIs, and the HIV RNA level was suppressed below 50 copies/ml at the next visit.

In the DRV/r monotherapy arm, 18 patients had sequential genotypes during sustained viremia, from 1 month to 36 months apart. The median number of minor IAS-USA PI mutations detected in the first genotype was 3 (range 2–4). Of these 18 patients 13 showed no change in the number and type of minor IAS-USA PI mutations over time (including three patients with no change from the sequence at baseline). Of the five patients with a change in sequence, one lost the L63P and V77I mutations, one lost the M36I mutation, one lost V82I but gained A71T, one lost K20R and gained V77I, and one gained G16E. Almost all the patients had HIV RNA levels below 50 copies/ml at baseline, so it was not possible to determine whether the minor IAS-USA PI mutations observed during the trial were treatment emergent or preexisting. Many of these minor IAS-USA mutations are commonly observed in samples from PI-naive patients in the Stanford HIV database.⁹ For example, the L63P mutation is detected in 55% of samples from PI-naive patients in the Stanford HIV database.⁹ In a previous publication we showed that the percentage of patients with these minor IAS-USA PI mutations did not differ between the DRV/r monotherapy and triple therapy arms of the MONET trial.¹⁰

There were three patients in the DRV/r monotherapy arm of the MONET trial who had genotypic resistance data at baseline (these were protocol violators with detectable HIV RNA levels at baseline) and during episodes of viremia during the trial. A summary of these three patients is shown in Table 1. Patient 1 had HIV RNA levels of 360–980 copies/ml around the week 60 visit, but then full suppression after intensification with tenofovir and FTC. Patient 2 had HIV RNA levels of 144, 585, and 69 copies/ml around the week 144 visit. Patient 3 had isolated blips in HIV RNA during the trial, but full suppression below 50 copies/ml at the end of the study with no change in treatment. However, there was no evidence for evolution of minor IAS-USA PI mutations in these patients, comparing the samples at baseline with those during the episodes of viremia.

Table 1.

Minor IAS-USA Mutations at Baseline and on Treatment for Patients Taking Darunavir/Ritonavir Monotherapy in the MONET Trial

Patient	Time point	HIV RNA (copies/ml)	Genotype
Patient 1	Baseline visit	278	I62V, L63P, V77I, I93L
	Week 84 visit	980	L63P, V77I, I93L
	Week 144	<50	n/a
Patient 2	Baseline visit	144	L63P, I64V
	Week 128 visit	144	I64V
	Week 144 visit	585	L63P, I64V
	Follow-up visit	69	L63P, I64V
Patient 3	Baseline visit	2500	L63P, A71T, V77I
	Week 3 visit	284	L63P, A71T, V77I
	Week 128 visit	264	L63P, A71T, V77I
	Week 144	<50	n/a

n/a, genotyping not possible because HIV RNA was undetectable at this patient visit.

After 380 patient-years of DRV/r monotherapy in the MONET trial, no patient has developed phenotypic resistance to darunavir. This analysis of all samples with HIV RNA >50 copies/ml is more detailed than most other trials, where only confirmed elevations above 400 copies/ml are tested for resistance. There has also been no evolution of new minor IAS-USA PI mutations after repeated genotyping in the MONET trial. In the MONOI trial of DRV/r monotherapy,¹¹ as well as the OK-04¹² and Kalesolo¹³ trials of lopinavir/ritonavir monotherapy, there has also been no evidence for treatment-emergent drug resistance when PI monotherapy is used as a switch option, for patients with HIV RNA <50 copies/ml at baseline. Only 62% of the samples showing detectable HIV RNA levels were successfully genotyped, but most patients showed only low-level viremia and genotypic resistance testing is less likely to be successful in samples with HIV RNA levels below 400 copies/ml.¹⁴

We did not evaluate changes in Gag cleavage sites during episodes of viremia in the MONET trial. In a similar trial of lopinavir/ritonavir for patients with HIV RNA <50 copies/ml at baseline (OK-04), mutations in Gag cleavage sites were observed both in the lopinavir/ritonavir monotherapy arm and the lopinavir/ritonavir plus 2NRTIs arm.¹⁵ This type of analysis needs to be repeated in a larger trial of DRV/r monotherapy, to evaluate the potential consequences for this treatment strategy.

Footnotes

Author Disclosure Statement

FP and JA were investigators in the MONET trial. CM is an employee of Janssen. AH has received consultancy payments from Janssen.

References

Gupta

, Hill

, Sawyer

et al. Emergence of drug resistance in HIV Type 1-infected patients after receipt of first-line highly active antiretroviral therapy: A systematic review of clinical trials. Clin Infect Dis, 2008; 47:712–722.

The UK Collaborative Group on HIV Drug Resistance and UK CHIC Study Group. Long term probability of detection of HIV-1 drug resistance after starting antiretroviral therapy in routine clinical practice. AIDS, 2005; 19:487–494.

Von Wyl

, Yerly

, Boni

, Burgisser

, Klimkait

, Battegay

et al. Swiss HIV Cohort Study. Emergence of HIV-1 drug resistance in previously untreated patients initiating combination antiretroviral treatment: A comparison of different regimen types. Arch Intern Med, 2007; 167:1782–1790.

Bierman

, van Agtmael

, Nijhuis

, Danner

, Boucher

. HIV monotherapy with ritonavir-boosted protease inhibitors: A systematic review. AIDS, 2009; 23:279–291.

Battegay

, Bucher

. Antiretroviral monotherapy: Should we abandon the principles of successful antiretroviral therapy? AIDS, 2010; 24,7:1057–1059.

Arribas

, Horban

, Gerstoft

et al. The MONET trial: Darunavir/ritonavir with or without nucleoside analogues, for patients with HIV RNA below 50 copies/ml. AIDS, 2010; 24,2:223–230.

Clumeck

, Rieger

, Banhegyi

, Schmidt

, Hill

, van Delft

et al. 96 week results from the MONET trial: A randomized comparison of darunavir/ritonavir with versus without nucleoside analogues, for patients with HIV RNA <50 copies/mL at baseline. J Antimicrob Chemother, 2011; 66,8:1878–1885.

Hirsch

, Gunthard

, Schapiro

et al. Antiretroviral drug resistance testing in adult HIV-1 infection: 2008 recommendations of an International AIDS Society-USA Panel. Clin Infect Dis, 2008; 47:266–285.

Stanford University HIV Drug resistance database: A curated public database designed to represent, store, analyse different forms of data underlying HIV drug resistance. http://hivdb.stanford.edu/index.html. 2010 January .

10.

Pulido

, Arribas

, Hill

, Van Delft

, Moecklinghoff

. Analysis of drug resistance during HIV RNA viraemia in the MONET trial of darunavir/ritonavir monotherapy. Antivir Ther, 2011; 16,1:59–65.

11.

Valentin

, Duvivier

, Lambert

, Flandre

, Cabie

, Molina

et al. Long-term efficacy of darunavir/ritonavir monotherapy in patients with HIV-1 viral suppression in the MONOI-ANRS 136 trial: Results at week 96. Presented at the Conference on Retroviruses and Opportunistic Infections (CROI), Boston, MA, February 2011[Abstr. 534].

12.

Pulido

, Perez-Valero

, Delgado

, Arranz

, Pasquau

, Portilla

et al. Risk factors for loss of virological suppression in patients receiving lopinavir/ritonavir monotherapy for maintenance of HIV suppression. Antivir Ther, 2009; 14:195–201.

13.

Meynard

, Buteloup

, Landman

, Bonnard

, Baillat

, Cabie

et al. Lopinavir/ritonavir monotherapy versus current treatment continuation for maintenance therapy of HIV-1 infection: The Kalesolo trial. J Antmicrob Chemother, 2010; 65:2436–2444.

14.

Prosperi

, Mackie

, Di Giambenedetto

, Zazzi

, Camacho

, Fanti

et al. Detection of drug resistance mutations at low plasma HIV-1 RNA load in a European multicenter cohort study. J Antimicrob Chemother, 2011; 66,8:1886–1896.

15.

McKinnon

, Delgado

, Pulido

, Shao

, Arribas

, Mellors

. Single genome sequencing of HIV-1 gag and protease inhibitor resistance mutations at virologic failure during the OK04 trial of simplified versus standard maintenance therapy. Antivir Ther, 2011; 16,5:725–732.