Kirby Institute for Infection and Immunity in Society, University of New South Wales, Sydney, Australia 3

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1 Antiviral Therapy 2011; 16: (doi: /IMP1906) Original article Etravirine and rilpivirine resistance in HIV-1 subtype CRF01_AE-infected adults failing non-nucleoside reverse transcriptase inhibitor-based regimens Torsak Bunupuradah 1,2,3, Jintanat Ananworanich 1,2,3,4, Ploenchan Chetchotisakd 5, Pacharee Kantipong 6, Supunnee Jirajariyavej 7, Sunee Sirivichayakul 4, Warangkana Munsakul 8, Wisit Prasithsirikul 9, Somnuek Sungkanuparph 10, Chureeratana Bowonwattanuwong 11, Virat Klinbuayaem 12, Kathy Petoumenos 2, Bernard Hirschel 13, Sorakij Bhakeecheep 14, Kiat Ruxrungtham 1,3 * 1 HIV-NAT, Thai Red Cross AIDS Research Centre, Bangkok, Thailand 2 Kirby Institute for Infection and Immunity in Society, University of New South Wales, Sydney, Australia 3 Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand 4 SEARCH, Thai Red Cross AIDS Research Centre, Bangkok, Thailand 5 Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand 6 Chiangrai Prachanukroh Hospital, Chiangrai, Thailand 7 Taksin Hospital, Bangkok, Thailand 8 BMA Medical College and Vajira Hospital, Bangkok, Thailand 9 Bamrasnaradura Institute, Nonthaburi, Thailand 10 Faculty of Medicine, Ramathibodi Hospital, Mahidol University, Bangkok, Thailand 11 Chonburi Hospital, Chonburi, Thailand 12 Sanpatong Hospital, Chiang Mai, Thailand 13 Geneva University, Geneva, Switzerland 14 The National Health Security Office, Bangkok, Thailand *Corresponding author kiat.r@chula.ac.th Background: We studied prevalence of etravirine (ETR) and rilpivirine (RPV) resistance in HIV-1 subtype CRF01_ AE infection with first-line non-nucleoside reverse transcriptase inhibitor (NNRTI) failure. Methods: A total of 225 adults failing two nucleoside reverse transcriptase inhibitors (NRTIs) plus 1 NNRTI in Thailand with HIV RNA>1,000 copies/ml were included. Genotypic resistance results and HIV-1 subtype were interpreted by Stanford DR database. ETR resistance was calculated by the new Monogram weighted score (Monogram WS; 4 indicating high-level ETR resistance) and by DUET weighted score (DUET WS; and 4 resulted in intermediate and reduce ETR response, respectively). RPV resistance interpretation was based on previous reports. Results: Median (IQR) age was 38 (34 42) years, 41% were female and CDC A:B:C were 22%:21%:57%. HIV subtypes were 96% CRF01_AE and 4% B. Antiretrovirals at failure were lamivudine (100%), stavudine (93%), nevirapine (90%) and efavirenz (10%) with a median (IQR) duration of 3.4 ( ) years. Median (IQR) CD4 + T-cell count and HIV RNA were 194 ( ) cells/mm 3 and 4.1 ( ) log 10 copies/ml, respectively. The common NNRTI mutations were Y181C (41%), G190A (22%) and K103N (19%). The proportion of patients with Monogram WS score 4 was 61.3%. By DUET WS, 49.8% and 7.5% of patients were scored and 4, respectively. Only HIV RNA 4 log 10 copies/ml at failure was associated with both Monogram WS 4 (OR 2.3, 95% CI ; P=0.003) and DUET WS 2.5 (OR 1.9, 95% CI ; P=0.02). The RVP resistance-associated mutations (RAMs) detected were K101P (1.8%), Y181I (2.7%) and Y181V (3.6%). All patients with RPV mutation had ETR resistance. No E138R/E138K mutations were detected. Conclusions: Approximately 60% of patients had highlevel ETR resistance. The role of ETR in second-line therapy is limited in late NNRTI failure settings. RVP RAMs were uncommon, but cross-resistance between ETR and RVP was high International Medical Press (print) (online) 1113

2 T Bunupuradah et al. Introduction Non-nucleoside reverse transcriptase inhibitor (NNRTI)-based HAART has been commonly prescribed in HIV-infected patients in resource-limited settings [1]. With the low genetic barriers for resistance evolution of NNRTI and limited access to HIV RNA monitoring and genotypic resistance testing, these patients are at high risk for developing extensive NNRTI and nucleoside reverse transcriptase inhibitor (NRTI) mutations at time of failure [2], which leads to limited options for the next regimens [3]. Etravirine (ETR), a second-generation NNRTI, formerly known as TMC-125, was recently approved for NNRTI-resistant adults if adequately supported by other active drugs in the second-line regimen [4,5]. The virological efficacy of ETR depends on the pattern and number of resistance-associated mutations (RAMs) in the HIV reverse transcriptase gene and its antiviral activity does not appear to be affected by isolated K103N mutation [4]. K103N is more commonly found in patients failing efavirenz (EFV) compared with those failing nevirapine (NVP) [6]. The tendency for mutation selection may differ between HIV subtypes [7]. In Thailand, the majority of HIV-infected individuals have HIV-1 subtype CRF01_AE [8], for which few reports on ETR resistance exist [9,10]. Rilpivirine (RPV; TMC278), another secondgeneration NNRTI, has recently been proven noninferior in efficacy to EFV in two Phase III studies [11]. E138, K101 and Y181, along with H221Y are mutations that confer RPV resistance. Azijn et al. [12] has recently reported their site-directed mutation study of RPV resistance and found that K101P, Y181I, Y181V and E138R were associated with median fold changes of 52, 15, 12 and 3.3, respectively. NNRTI-based HAART is the first-line therapy in the National HIV Treatment Guidelines [13]. The most commonly used first-line regimen in Thailand is a fixed dose combination of stavudine (d4t) plus lamivudine (3TC) plus NVP. Since 2007, the Thai National Health Security Office (NHSO) has provided free HIV RNA testing every 12 months and genotypic resistance testing when indicated. The objectives for this report are to explore the possible role of ETR and RPV in second- line antiretroviral therapy in Thai patients with predominantly CRF_01AE infection who are failing NNRTI-based regimens, and to identify the predicting factors for ETR mutations. Methods From May 2008 to November 2009, Thai HIV-infected adults failing NNRTI-based regimens from nine referral hospitals were enrolled in a multicentre trial, HIV STAR study (clinical trial number NCT ; Additional file 1). The inclusion criteria were HIV-infected adults, aged 18 years, on NNRTI (NVP or EFV)-based HAART for at least 6 months, plasma HIV RNA 1,000 copies/ml, and had never used protease inhibitors (PIs). The study protocol was approved by all local and the Thai Ministry of Public Health ethics committees. All enrolled HIV-infected adults gave informed consent. Complete blood count, CD4 + T-cell count and HIV RNA at virological failure while patients were taking NNRTI-based HAART were tested by the routine laboratory service in the hospitals. All genotypic resistance testing were performed centrally at the Vaccine and Cellular Immunology (VCI) Laboratory (Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand) [14,15]. The VCI Laboratory has participated in the TreatAsia Quality Assurance Scheme (TAQAS) since 2006 [15]. The mutations were defined according to the Stanford Interpretation System. Multi-NRTI mutations were defined as having thymidine analogue mutations (TAMs) 4 or Q151M complex or 69 insertion. Etravirine resistance ETR resistance was evaluated using two algorithms: Monogram weighted score (Monogram WS) [16 19] and DUET weighted score (DUET WS) [16,20,21]. Monogram WS Five mutations had a weighted score of 4: L100I, K101P, Y181C/I/V. Mutations with a weighted score of 3 were E138A/G, V179E, G190Q, M230L and K238N. Mutations with a weighted score of 2 were K101E, V106A/I, E138K, V179L, Y188L and G190S. The mutations that scored 1 were V90I, A98G, K101H, K103R, V106M, E138Q, V179D/F/I/M/T, Y181F, V189I, G190A/E/T, H221Y, P225H and K238T [16 19]. Having a weighted score of 4 was associated with reduced in vitro susceptibility of ETR. DUET WS Briefly, each of the NNRTI mutations (a total of 17 ETR RAMs) were weighted: 3.0 (Y181I/V), 2.5 (L100I, K101P, Y181C and M230L), 1.5 (V106I, V179F, E138A and G190S) or 1.0 (V90I, A98G, K101E/H, V179D/T and G190A). The total DUET WS of 0 2, and 4 resulted in highest response (74%), intermediate response (52%) and reduced response (38%) to ETR, respectively [16,20,21]. Rilpivirine resistance-associated mutations We also identified three mutations (K101P, Y181I and Y181V) that were recently reported to have decreased in vitro susceptibility to the new NNRTI, RPV (TMC278), and to ETR [22]. Moreover, E138R was recently identified as a new NNRTI RAM that International Medical Press

3 Etravirine and rilpivirine resistance in first-line NNRTI-failure Table 1. Baseline characteristics of Thai HIV-infected adults who failed NNRTI-based HAART Total adults Adults with Monogram Adults with Monogram Characteristic (n=225) weighted score <4 (n=87) weighted score 4 (n=138) Median age, years (IQR) 38 (34 42) 38 (34 44) 37 (34 42) Female, % CDC clinical classification, A:B:C % 22:21:57 27:15:58 19:24:57 Median weight, kg (IQR) 57.7 (52 65) 58.7 (52 64) 57.5 ( ) Mode of HIV transmission Heterosexual, % Homosexual, % Intravenous drug user, % Tattoo, % Unknown, % Median CD4 + T-cell count before HAART, cells/mm 3 (IQR) 43 (15 108) 44 (16 120) 42 (15 106) Median CD4 + T-cell count nadir, cells/mm 3 (IQR) 51 (14 110) 52 (13 100) 50 (14 117) Median current haemoglobin, g/dl (IQR) 13 ( ) 13 ( ) 12.9 ( ) Median current CD4 + T-cell count, cells/mm 3 (IQR) 194 ( ) 195 ( ) 190 ( ) Median current HIV RNA, log 10 copies/ml (IQR) 4.1 ( ) 3.9 ( ) 4.3 ( ) a Nevirapine:efavirenz, % 90:10 95:5 86:14 Median duration of NNRTI-based HAART, years (IQR) 3.4 ( ) 3.6 ( ) 3.4 ( ) Mean number of NRTI mutations (±sd) 2.6 (2.0) 1.9 (2.0) 3.0 (1.9) The current haemoglobin, CD4 + T-cell count and HIV RNA were measured at virological failure. a P= NNRTI, non-nucleoside reverse transcriptase inhibitor; NRTI, nucleoside reverse transcriptase inhibitor. was always present in combination with other NNRTI RAMs, such as L100I and M230I. Together these induced high levels of resistance to RPV, ETR and EFV [12,23]. Recently, E138K was reported in a substantial number of patients failing to RPV [23]. We also determined whether our subjects had the combination of NNRTI RAMs that were associated with high fold changes in vitro antiviral activity of RPV, EFV and ETR, namely K103N plus Y181I, L100I plus K103N plus Y181C, K101P plus K103N plus V108I and V179F plus Y181C plus F227C [12]. Statistical analysis Descriptive statistics (medians, interquartile ranges, means, standard deviations and percentages) were used to summarize demographic and clinical characteristics of the HIV-infected participants. The agreement of the new Monogram WS 4 and DUET WS 2.5 was calculated by kappa statistic [24], for example, kappa represents the substantial agreement and represents almost perfect agreement. Logistic regression analysis was used to determine predicting factors of Monogram WS 4 and DUET WS 2.5. Potential risk factors were entered into a binary logistic regression model. Forward stepwise methods were used to develop multivariate models. Variables with P-value <0.10 in the univariate analyses were considered for inclusion in the multivariate analyses. Analyses were performed in Stata (version 10.0; Stata corp., College Station, TX, USA). Results A total of 225 HIV-infected Thai adults were enrolled. The median (IQR) age was 38 (34 42) years, 41% were female and the CDC clinical classification A:B:C were 22%:21%:57%. The median (IQR) current CD4 + T-cell count and HIV RNA were 194 ( ) cells/mm 3 and 4.1 ( ) log 10 copies/ml, respectively (Table 1). HIV subtype CRF01_AE was 96% and subtype B was 4%. Median (IQR) first-line HAART duration was 3.4 ( ) years. All patients (100%) used 3TC. d4t, zidovudine, tenofovir (TDF) and abacavir were used as the backbone NRTIs in 93%, 5%, 1% and 0.5%, respectively. With regards to NNRTIs, NVP and EFV were used in 90% and 10%, respectively. The most common NRTI mutations were D67N (30.2%), K70R (22.2%) and T215F (15.6%). A total of 177 (78.7%) adults had M184V and 14 (6.2%) had K65R (Table 2). Multi-NRTI mutations were 4 TAMs in 31% and Q151M in 6%; none had 69i. The most common NNRTI mutations were Y181C (40.9%), G190A (22%) and K103N (19%). However, when only NNRTI mutations listed in Monogram WS was considered, the most common NNRTI mutations were Y181C (40.9%), H221Y (14.7%) and K101E (14.2%). ETR resistance According to the new Monogram WS algorithm, percentages with a score of 0 was 16.4%, score 1 was Antiviral Therapy

4 T Bunupuradah et al. 8.0%, score 2 was 9.0%, score 3 was 5.3% and score 4 was 61.3%. The range for Monogram WS was from 0 to 9. Adults with Monogram WS<4 had a significantly higher proportion of K65R and F116Y compared with adults with score 4 (Table 2). For NNRTI mutations, more adults with Monogram WS 4 had these three NNRTI mutations (V179D [P=0.03], V179I [P=0.04] and H221Y [P<0.0001]) compared with adults with score <4 (Table 3). According to the DUET WS algorithm, 96 (42.7%), 112 (49.8%) and 17 (7.5%) of HIV-infected adults were scored and 4, respectively. The kappa statistic of Monogram WS 4 and DUET WS 2.5 was Predicting factor of ETR resistance with Monogram WS By multivariate logistic regression analysis, HIV RNA 4 log 10 copies/ml at time of failure was associated with new Monogram WS 4 (OR 2.3, 95% CI ; P=0.003). However, age, gender, body weight, CDC clinical classification, CD4 + T-cell count, NNRTI type and HAART duration did not predict ETR score 4 (Table 4). Predicting factor of ETR resistance with DUET WS Factors associated with DUET WS 2.5 was HIV RNA 4 log 10 copies/ml at time of failure (OR 1.9, 95% CI ; P=0.02). Age, gender, body weight, CDC clinical classification, CD4 + T-cell count, NNRTI type and HAART duration were not associated with DUET WS 2.5 (Table 5). RPV resistance-associated mutations The number of adults (%) with mutations associated with resistance to RPV (TMC278) were 4 (1.8%) for K101P, 6 (2.7%) for Y181I and 8 (3.6%) for Y181V. All of these adults with RPV RAM had ETR resistance both by Monogram WS 4 and DUET WS 2.5. No E138R or E138K were identified. One (0.4%) patient had the combined mutations of L100I plus K103N plus Y181C. None had a combination of RAMs that have been shown to associated with a high increase of fold change in susceptibility to RPV, that is, K103N plus Y181I or K101P plus K103N plus V108I or V179F plus Y181C plus F227C. Discussion High levels of ETR resistance were seen in approximately 60% of our patients, of whom the majority had failed d4t/3tc/nvp first-line treatment after being on it for about 3 years. The background NRTI resistance was also common with almost all patients having 3TC resistance, about 40% having multi-nrti resistance Table 2. Nucleoside reverse transcriptase mutations in 225 adults failing NNRTI-based HAART All 225 adults, 87 Adults with Monogram 138 Adults with Monogram NRTI mutations n (%) weighted score <4, n (%) weighted score 4, n (%) P-value M41L 33 (14.7) 9 (10.3) 24 (17.4) 0.15 E44D 7 (3.1) 2 (2.3) 5 (3.6) 0.58 A62V 10 (4.4) 5 (5.8) 5 (3.6) 0.45 K65R 14 (6.2) 11 (12.6) 3 (2.2) D67N 68 (30.2) 25 (28.7) 43 (31.2) 0.70 T69D 8 (3.6) 2 (2.3) 6 (4.4) 0.42 T69N 22 (9.8) 11 (12.6) 11 (8.0) 0.25 K70E 1 (0.4) 0 1 (0.7) 0.43 K70R 50 (22.2) 24 (27.6) 26 (18.8) 0.12 L74V 9 (4.0) 2 (2.3) 7 (5.1) 0.30 V75I 14 (6.2) 8 (9.2) 6 (4.4) 0.14 F77L 8 (3.6) 4 (4.6) 4 (2.9) 0.50 Y115F 6 (2.7) 3 (3.5) 3 (2.2) 0.56 F116Y 9 (4.0) 7 (8.1) 2 (1.5) 0.01 V118I 20 (8.9) 6 (6.9) 14 (10.1) 0.40 Q151M 13 (5.8) 7 (8.1) 6 (4.4) 0.25 M184V 177 (78.7) 69 (79.3) 108 (78.3) 0.85 L210W 24 (10.7) 9 (10.3) 15 (10.9) 0.90 T215F 35 (15.6) 11 (12.6) 24 (17.4) 0.34 T215Y 24 (10.7) 9 (10.3) 15 (10.9) 0.90 K219E 16 (7.1) 8 (9.2) 8 (5.8) 0.33 K219Q 30 (13.3) 11 (12.6) 19 (13.8) 0.81 NNRTI, non-nucleoside reverse transcriptase inhibitor International Medical Press

5 Etravirine and rilpivirine resistance in first-line NNRTI-failure and 6% having TDF resistance; this greatly hinders the likelihood of virological success of using ETR with TDF and 3TC, the most likely NRTI second-line option in Thailand and most resource-limited settings. Even with a PI in the second-line regimen, ETR would only benefit approximately 40% of the patients. RPV RAMs were uncommon but a high degree of cross-resistance between ETR and RPV was found. The prevalence of ETR and RPV mutations is probably underestimated due to the fact that no genotypic resistance tests have been done for patients failing treatment with HIV-1 RNA between 50 and 1,000 copies/ml. Failing the d4t/3tc/nvp regimen is associated ETR mutations. Kiertiburanakul et al. [10] reported that 33% of 184 Thai HIV-infected adults who had mainly used d4t/3tc/nvp and who had experienced virological failure had a reduced response to ETR. Half of the 91 Nigerian adults infected with non-b subtype HIV-1 Table 3. Non-nucleoside reverse transcriptase mutations stratified by new Monogram weighted score in 225 adults failing NNRTI-based HAART 87 Adults with new Monogram 138 Adults with new Monogram NNRTI mutations All 225 adults, n (%) weighted score <4, n (%) weighted score 4, n (%) P-value Score 4 L100I 2 (0.9) 0 2 (1.5) 0.23 K101P 4 (1.8) 0 4 (2.9) 0.11 Y181C 92 (40.9) 0 92 (66.7) < Y181I 6 (2.7) 0 6 (4.4) Y181V 8 (3.6) 0 8 (5.8) 0.02 Score 3 E138A 5 (2.2) 0 5 (3.6) 0.07 E138G 2 (0.9) 0 2 (1.5) 0.23 V179E 1 (0.4) 0 1 (0.7) 0.43 G190Q 0 (0) M230L 4 (1.8) 1 (1.2) 3 (2.2) 0.57 K238N 0 (0) Score 2 K101E 32 (14.2) 9 (10.3) 23 (16.7) 0.19 V106A 3 (1.3) 1 (1.2) 2 (1.5) 0.85 V106I 0 (0) E138K 0 (0) V179L 2 (0.9) 0 2 (1.5) 0.23 Y188L 13 (5.8) 8 (9.2) 5 (3.6) 0.08 G190S 4 (1.8) 1 (1.2) 3 (2.2) 0.57 Score 1 V90I 12 (5.3) 2 (2.3) 10 (7.3) 0.11 A98G 20 (8.9) 5 (5.6) 15 (10.9) 0.19 K101H 2 (0.9) 1 (1.2) 1 (0.7) 0.74 K103R 3 (1.3) 0 3 (2.2) 0.17 V106M 1 (0.4) 0 1 (0.7) 0.43 E138Q 8 (3.6) 5 (5.8) 3 (2.2) 0.16 V179D 7 (3.1) 0 7 (5.1) 0.03 V179F 0 (0) V179I 47 (20.9) 12 (13.8) 35 (25.4) 0.04 V179M 0 (0) V179T 4 (1.8) 2 (2.3) 2 (1.5) 0.64 Y181F 0 (0) V189I 3 (1.3) 0 3 (2.2) 0.17 G190A 49 (21.8) 20 (23.0) 29 (21.0) 0.73 G190E 0 (0) G190T 0 (0) H221Y 33 (14.7) 3 (3.5) 30 (21.7) < P225H 5 (2.2) 3 (3.5) 2 (1.5) 0.32 K238T 1 (0.4) 0 1 (0.7) 0.43 NNRTI, non-nucleoside reverse transcriptase inhibitor. Antiviral Therapy

6 T Bunupuradah et al. Table 4. Factors associated with new Monogram weighted score 4 Univariate logistic regression Multivariate logistic regression Factors OR (95% CI) P-value OR (95% CI) P-value Age, years 0.9 ( ) 0.26 Sex Male 1 Female 1.1 ( ) 0.79 CDC clinical classification A 1 B 2.3 ( ) 0.07 C 1.4 ( ) 0.34 Mode of HIV transmission Heterosexual 1 Homosexual 1.3 ( ) 0.66 Intravenous drug user 1.3 ( ) 0.82 Tattoo 0.7 ( ) 0.77 Unknown 1.2 ( ) 0.57 Body weight, kg 1.0 ( ) 0.98 CD4 + T-cell before NNRTI-based HAART <50 cells/mm cells/mm ( ) 0.85 CD4 + T-cell nadir <50 cells/mm cells/mm ( ) 0.93 Current haemoglobin <10 g/dl 1 10 g/dl 1.6 ( ) 0.42 Current CD4 + T-cell count <200 cells/mm cells/mm ( ) 0.68 Current HIV RNA <4 log 10 copies/ml 1 4 log 10 copies/ml 2.3 ( ) ( ) Duration of first-line NNRTI HAART <3.5 years years 1.0 ( ) 0.94 NNRTI Efavirenz 1 Nevirapine 0.4 ( ) 0.09 NNRTI, non-nucleoside reverse transcriptase inhibitor. and failing NVP-based therapy had high levels of ETR resistance [25]. In a study of Thai HIV-infected children failing either NVP or EFV-based HAART [17], almost half had high levels of ETR resistance. The use of NVP was associated with Monogram WS 4 [10,26]. A study has confirmed worse response to ETR after failing NVP compared with EFV [27]. The lack of an association between high levels of ETR resistance and NVP use in our study is likely explained by the fact that almost all patients used NVP. The predicting factors for unlikely to benefit from ETR was HIV RNA 4 log 10 copies/ml at time of failure. High levels of HIV RNA is a known driving force for resistance accumulation [17,28]. Our patients had access to at most once yearly HIV RNA testing. More frequent HIV RNA monitoring may have detected virological failure earlier and limited the resistance mutations. Compared with studies in resource-rich settings with more frequent HIV RNA testing, 67.2% and 67.5% of 5,482 samples from HIV-infected individuals were classified as genotypic susceptible to ETR using Monogram WS and DUET WS, respectively [29]. As genotypic resistance testing is required before prescribing ETR to any HIV-infected patients, it further limits the use of ETR in resource-limited settings. The common ETR mutations in our patients were Y181C, H221Y and K101E, which were similar to common ETR mutations in patients with HIV subtype CRF_01 AE and in other non-b subtypes [10,25]. Evaluation of ETR susceptibility from genotypic testing results can use the different interpretation International Medical Press

7 Etravirine and rilpivirine resistance in first-line NNRTI-failure Table 5. Factors associated with DUET weighted score 2.5 Univariate logistic regression Multivariate logistic regression Factors OR (95% CI) P-value OR (95% CI) P-value Age, years 1.0 ( ) 0.31 Sex Male 1 Female 1.7 ( ) 0.07 CDC clinical classification A 1 B 2.9 ( ) 0.03 C 1.3 ( ) 0.55 Mode of HIV transmission Heterosexual 1 Homosexual 0.7 ( ) 0.60 Intravenous drug user 1.5 ( ) 0.76 Tattoo 0.7 ( ) 0.83 Unknown 1.0 ( ) 0.98 Body weight, kg 1.0 ( ) 0.07 CD4 + T-cell count before NNRTI-based HAART <50 cells/mm cells/mm ( ) 0.43 CD4 + T-cell count nadir <50 cells/mm cells/mm ( ) 0.46 Current haemoglobin <10 g/dl 1 10 g/dl 1.7 ( ) 0.35 Current CD4 + T-cell count <200 cells/mm cells/mm ( ) 0.99 Current HIV RNA <4 log 10 copies/ml log 10 copies/ml 1.9 ( ) ( ) 0.02 Duration of first-line NNRTI HAART <3.5 years years 1.0 ( ) 0.92 NNRTI Efavirenz 1 Nevirapine 0.8 ( ) 0.57 NNRTI, non-nucleoside reverse transcriptase inhibitor. systems. In this report, two algorithms were used. Monogram WS was proposed by Monogram Company researchers based on in vitro and ETR fold changes [18]. DUET WS was based on the DUET studies [21,30,31]. We found the inter-observer agreement, the kappa statistic, between Monogram WS 4 and DUET WS 2.5 to be almost perfect [24]; therefore, we feel that either algorithm can be used interchangeably. The association of ETR and phenotypic susceptibility has been reported. Picchio et al. [29] reported that 99.5% of 9,458 samples with the NNRTI genotype susceptible to ETR, calculated by Monogram WS and DUET WS, had phenotypic susceptibility to ETR (Antivirogram fold change 3). This data supports clinical implication of using Monogram WS or DUET WS in resource-limited settings. One factor that is associated with better virological response to ETR in NNRTI-experienced patients was the number of new antiretrovirals, including darunavir, raltegravir and T20, in the subsequent regimen [27,31 34]. A previous study had shown that using ETR with two NRTIs in patients failing NNRTI was unsuccessful compared with using a boosted PI with two NRTIs even when there were comparable NRTI mutations and an optimal of two NRTIs backbone [35]. Our study also confirms the limited use of ETR in this setting. Unfortunately, darunavir and the other new drugs are expensive and not widely accessible in resource-limited settings. Using ETR with lopinavir/ritonavir would be beneficial Antiviral Therapy

8 T Bunupuradah et al. in patients without high levels of ETR resistance; however, only one report has been recorded [36]. Only a small percentage had RPV resistance, making this a viable option for our patients. However, the efficacy of RPV has been studied only in antiretroviral-naive HIV-infected adults in clinical trial Phase III [37]. There are limited data on RPV mutations because it is a new drug. Whether this new NNRTI may be beneficial as a treatment option after NVP/EFV-based first-line failure is unknown and is warranted for further investigation. In conclusion, in a predominantly NVP treated cohort of Thai patients with treatment failure after 3.5 years of first-line HAART, only approximately 60% would benefit from ETR in their next regimen. Patients with high HIV RNA levels were more likely to have high levels of ETR resistance. The usefulness of ETR in second-line therapy is limited in resource-limited settings where HIV RNA monitoring is infrequent and extensive NRTI/NNRTI resistance occurs. Moreover, the need for genotyping to guide the selection of ETR further limits its use. The RPV RAMS were uncommon but high degree of cross-resistance with ETR was found. Further investigations are needed to assess the use of RPV in patients failing NNRTI-based HAART. Acknowledgements The HIV Second-line Therapy AntiRetroviral (HIV STAR; Additional file 1) study in patients who failed NNRTI-based regimens (clinical trial number NCT ) was supported by grants from the Thai NHSO, Swiss cohort study and the National Research Council of Thailand (NRCT). The antiretrovirals and laboratory monitoring were provided by the Thai NHSO. We are grateful to the patients for their participation in this study. We thank the Program for HIV Prevention and Treatment (PHPT) laboratory for facilitating laboratory testing and sample shipment of study sites in the North of Thailand. We thank the HIV STAR study team for their dedication to this study. The preliminary analysis of this study has been presented as a poster (MOPDB104) at the XVIII International AIDS Conference (18 23 July 2010, Vienna, Austria). Disclosure statement KR has been participated at Tibotec scientific advisory committee meetings and has been supported for two international AIDS conferences by Janssen-Cilag (Thailand). KR has also received a Professional Researcher Strengthen Grant from the National Science and Technology Development Agency (NSTDA), Biotec and the Ministry of Science and Technology. 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