HIV-1 Protease Inhibitors Resistance Profiles in Patients with Virological Failure on LPV/r-based 2nd Line Regimen in Cambodia

ISSN:2155-6113 JAR, an open access journal J AIDS Clinic Res Drug Resistance: HIV HIV-1 Protease Inhibitors Resistance Profiles in Patients with Virological Failure on LPV/r-based 2nd Line Regimen in Cambodia Eric Nerrienet1*, Janin Nouhin1, Sopheak Ngin1, Olivier Segeral2,3, Sreymom Ken1, Kerya Phon1, Chanroeurn Hak2, Vara Ouk2, Vonthanak Saphonn4, Laurent Ferradini5 and Jean-Francois Delfraissy3,6 1HIV/Hepatitis Laboratory, Pasteur Institute, Phnom Penh, Cambodia 2ESTHER/Calmette hospital, Phnom Penh, Cambodia 3Clinical Immunology Department, Bicetre Hospital, Kremlin Bicetre, France 4National Center for HIV/AIDS and STD (NCHADS), Phnom Penh, Cambodia 5FHI 360 Cambodia, Phnom Penh, Cambodia 6ANRS, Paris, France


Introduction
HAART treatment programs have proven the feasibility and efficacy of first line antiretroviral (ARV) regimen in resource-poor settings to be similar to those reported in developed countries [1,2]. In Cambodia, 13.6 million inhabitants, the number of HIV-infected patients on reverse transcriptase inhibitors (RTIs)-based first line ARV regimen increased dramatically these last few years, reaching nearby 41,660 by the end of 2010 [3]. Favourable outcomes were reported in Cambodia among adults and children after one to four years of first line ARV regimen associating AZT or D4T, 3TC/FTC, NVP or EFV [4][5][6][7]. At the same time, highly active antiretroviral therapy (HAART) duration is increasing and treatment failures become more common leading to a rising number of patients initiated on second line protease inhibitor (PI)-based HAART regimen. As of end of 2010, the National Centre for HIV/AIDS, Dermatology and STD (NCHADS) reported that nearly 1,800 patients already switched to a protease inhibitor (PI)based 2 nd line in Cambodia [8].
We recently reported that ritonavir boosted Lopinavir (LPV /r )-based second line ARV regimen in Phnom Penh was associated with a high rate of virological suppression and good immune reconstitution after 24 months of treatment [10].
As expected by the rising number of patients on LPV /r -based 2 nd line regimen and the extended duration of follow-up on 2 nd line regimen, the emergence of 2 nd line treatment failures is becoming an important challenge in developing countries with limited virological follow-up and no optional third line regimen [11]. Indeed, mainly because of their price, the number of PI available in these countries remains limited and no integrase inhibitors-based 3 rd line ARV regimens are currently available.
line regimen). Such information might help to estimate the need for alternative regimen in Cambodia and to design appropriate alternative protease inhibitor (PI)-based 2 nd line or integrase inhibitor (II)-based 3 rd line regimen in Cambodia.

Patients and plasma specimen
By the end of October 2010, 1,888 blood specimens have been referred for ARV resistance testing as routinely performed at Pasteur Institute of Cambodia (IPC). The nature and the duration of the PIbased therapy were collected and recorded along with the samples. Among 130 patients on LPV/r-based second line regimen, 89 experienced virological failure (VL > 250 copies/ml) after at least 6 months on LPV /r -based 2 nd line regimen. Fifty percent of them were patients followed-up by non governmental organizations (NGOs), 30% were referred by the private sector, and 20% by national hospitals. Most of them are regularly monitored for HIV VL and resistance testing at IPC.

HIV RNA Viral load (VL)
Plasma samples were used for HIV RNA viral load (VL) assessment as well as drug resistance genotyping. HIV RNA was extracted from plasma using QIAamp Viral RNA mini kit (Qiagen, Hilden, Germany). The HIV RNA VL was assessed using the G2 Generic HIV-1 viral load ANRS kit (Biocentric, Bandol, France) [12,13]. Using 0.2 ml of plasma, the threshold of the assay was 250 copies/ml.

HIV Drug Resistance Analysis
Bulk sequencing of protease (PR), reverse transcriptase (RT) and integrase (IN) genes were performed according to the complete sequencing procedures and primer sequences previously described [14] in order to detect PI, RTI and II resistance associated mutations (RAMs).
Briefly, nested-PCR amplified fragments were sent to the Macrogen Company (Macrogen Inc., Seoul, Republic of Korea) and chromatograms sent electronically to the Pasteur Institute of Cambodia were verified, analyzed, and interpreted using Ceq2000 (Beckman Coulter) software. Nucleotides and amino acid sequences were compared with the HXB2 reference sequence using Mega 4 [15]. Drug resistance mutations were defined according to the last updated ANRS algorithms [14] taking into account the natural genetic polymorphism of CRF01_AE viruses predominant in South East Asia. HIV subtypes were determined by sequence analysis of amplified fragments using CLUSTAL X 1.81 software [16] and subtype reference sequences set from Los Alamos HIV sequence Database.

Nucleotide accession numbers
Nucleotide sequences of HIV-1 PR, RT, and IN obtained from patients have been submitted to GenBank under the accession numbers JN675743 to JN675813, JN675814 to JN675896, and JN675719 to JN675742, respectively.

First group of patients with lack of HIV-protease gene amplification (group A)
Three groups of patients could be defined according to their distinct pattern of resistance in the HIV protease genes. The HIV protease gene could not be PCR amplified for 18 (16 males and 2 females) of the 89 patients (20.2%, referred thereafter as group A). Their median duration on LPV /r -based 2 nd line regimen was 18 months [IQR: 13-27; range 6-45] and the median HIV RNA VL was at 2.9Log 10 copies/ml [IQR: 2.8-3.4; range 2.6-4.1] ( Table 1). The most common LPV/r based 2 nd line regimen associated TDF or ddI plus 3TC (n=11) or ABC plus DDI (n=3).

Third group of patient with high level of resistance to PIs (group C)
A third group of patients (n=29, 32.6%) presented PI-RAMs conferring resistance to at least one PI (thereafter referred as Group C). The median age in this group was 40 years [IQR: 36-42, range 9-55], the median duration on LPV /r -based 2 nd line regimen was 35 months [IQR: 24-60, range 9-144] and the median VL was 5Log 10 copies/ml [IQR: 4.3-5.4; range: 2.5-6.3] ( Table 1). In contrast with what was observed for the other groups A and B, a large majority of males (n=27, 93.1%) was found in this group. Compared with the previous two groups, the median duration on LPV/r was significantly higher (p=0.0001) ( Table 1).

Discussion
We report here the antiretroviral drug resistance profiles of patients presenting detectable viral load after at least 6 months on LPV /r -based 2 nd line therapy. The first major finding was that no PI resistance was observed for almost two-third of the 89 patients studied outlining the importance of adherence strengthening before envisaging any costly treatment modifications as recently reported [25].
Indeed, the fact that HIV-1 protease PCR amplifications were unsuccessful for 20% of the patients (group A) could be explained by their low level of VL (< 1,000 copies/ml, Table 1) below the sensitivity (1000 copies/ml) of the technique used [14] In the group B (47.2% of the patients) with no resistance to PIs, some patients had viruses with PI-RAMs which, however, did not confer resistances to PI according to HIV-1 B subtype interpretation algorithms. Even we cannot completely exclude the presence of PI-RAMs in viruses infecting patients of group A, these results suggest that wild type or quasi wild type viruses might have emerged in these patients probably because of recent treatment discontinuation or sub-optimal adherence. All together, these 2 groups of patients with no resistance to PIs represented more than 2/3 (67.4%) of patients studied. Interestingly, controlled viral loads performed 6 to 9 months after this first detectable VL revealed that strengthening adherence alone lead to the control of HIV replication for 80% of them (data not shown).
Thus, in Cambodian settings, where few 2 nd line alternatives and no 3 rd line regimen are available, focusing on adherence strengthening for patients on 2 nd line regimen is critical with a control of the viral load after few months following LPV/r initiation (to detect early poor adherence to 2 nd line).
On the other hand, a third group (Group C) representing almost one-third (32.6%) of the patients displayed multiple resistances to PIs and clearly required urgent treatment modifications. In depth clinical investigations obtained for some of them revealed they all had experienced multiple PI drugs distinct from LPV /r -more than 10 years ago which might have favored the emergence of multiple PI resistance over time. This is similar to the results of first line treatment failures mostly occurring in ARV non-naïve patients in Cambodia [4,26]. In addition, the significantly longer period of follow-up on LPV /r -based As expected, almost all of the patients of the group C were resistant to LPV /r , the PI back-bone of 2 nd line regimen in Cambodia. Patients of group C were also found resistant to many other PIs with the highest number of patient (96.5%) resistant to IDV. Previous exposure to IDV but also the emergence of cross-reacting mutations, such as M46I/L, V82A/F/M/S/T and I84A/V, under the selective pressure of LPV /r might explain such a high level of resistance to IDV. Indeed, IDV together with SQV /r and NFV were easily available in the late 90s and often prescribed privately or self administered as initial ARV treatment or after first line failure in Cambodia. Many of these patients presented also resistances to other PIs like ATV/r, FPV/r, NFV, SQV/r or DRV/r often to 3 or 4 of them at the same time. Similarly to IDV resistance, such a high level of resistance to several PIs might have been cross-selected under extended LPV /r exposition and also through previous exposure to mostly NFV and/or SQV/r since other PIs including ATV/r, FPV/r, or DRV/r were and are still not or poorly available in the region.
Only 24.1% of patients from the group C were found resistant to DRV /r ( Figure 1B), including 5/7 patients only possibly resistant. Unfortunately, the ARV histories and the small number of patients followed did not allow us to identify risk factors for development of resistance to DRV /r . Even some of the patients still sensitive to DRV /r , have already selected one or two DRV-RAMs, the high level of sensitivity (over 75%) observed to DRV /r made it a good candidate for an alternative ARV regimen for this group of patient.
In order to better figure out the type of potential regimen to be proposed to already PI resistant patients, genotyping analysis of the RT and IN genes gave us more information about still available backbone ARV drugs. ETV was the RTI with the lowest level of resistance even with almost 45% of the patients resistant or possibly resistant, compared to the 25 to 30% recently reported among HIV-1 infected patients failing first and second line treatment in Mali [27]. It should be also added that, among our patients still sensitive to ETV, some already harbored ETV-RAMs which could jeopardize the future virological efficacy of an alternative therapy combining ETV.
For these patients under confirmed 2 nd line treatment failure (group C) an alternative ARV regimen should be considered case per case considering each treatment history. However, if we consider the possibility of a true 3 rd line regimen at least, two sensitive ARV drugs should be considered including a new class of ARV, like the integrase inhibitors. As previously reported [29], the combination of DRV/ RAL and potentially ETV could be considered as a 3 rd line option. Interestingly, 2 patients (#28 and #29, Figure 3) who started ETV/DRV/ RAL therapy had an undetectable VL after 3 to 4 months later.
For such patients who currently need to be switched from their 2 nd line regimen, the poor availability of alternative ARV class drugs such as ETV/DRV/RAL and its extremely high cost (2000 USD/month) in Cambodia remain dramatic barriers for their survival.
Most of the 29 patients studied here have been previously exposed to several RTI and PI probably at the beginning of the epidemic in Cambodia when ARVs were not yet officially available. It is possible that less people will truly fail to the current 2 nd line regimen since the vast majority of patients currently initiated on such regimen through the National Program in Cambodia were ARV-naive before starting first line regimen and probably were never exposed to PIs. This will have to be confirmed by further prospective long term evaluation of 2 nd line efficacy in Cambodia.