Journal of Antimicrobial Chemotherapy Advance Access published August 21, 2012

J Antimicrob Chemother doi:10.1093/jac/dks331

Nevirapine use, prolonged antiretroviral therapy and high CD4 nadir values are strongly correlated with undetectable HIV-DNA and -RNA levels and CD4 cell gain
Loredana Sarmati1*, Saverio Giuseppe Parisi2, Marco Montano1, Samantha Andreis2, Renzo Scaggiante3, Andrea Galgani4, Magdalena Viscione1, Gaetano Maffongelli1, Alessandra Ricciardi1, Carolina Andreoni5, ` Stefano Boros6, Giorgio Palu2 and Massimo Andreoni1
1 Clinical Infectious Diseases, Tor Vergata University, Rome, Italy; 2Department of Histology, Microbiology and Medical Biotechnology, University of Padua, Padua, Italy; 3Infectious Diseases Unit, Padua Hospital, Padua, Italy; 4Interdepartmental Centre-Animal Technology Station, Tor Vergata University, Rome, Italy; 5Clinical Infectious Diseases, La Sapienza University, Rome, Italy; 6Department of `, Immuno-mediated Infectious Diseases, Istituto Superiore di Sanita Rome, Italy

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*Corresponding author. Tel: +390672596874; Fax: +390672506873; E-mail: sarmati@med.uniroma2.it

Received 24 June 2012; returned 13 July 2012; revised 19 July 2012; accepted 21 July 2012 Objectives: To evaluate the correlations of the combination of undetectable HIV-DNA (,10 copies/106 peripheral blood mononuclear cells) and HIV-RNA (,1 copy/mL of plasma) levels and a CD4 cell count of .500 cells/mm3 (dened as the treatment goal) in a group of 420 antiretroviral treatment (ART) responder patients. Methods: A cross-sectional, open-label, multicentre trial was conducted in a cohort of 420 HIV-infected ARTtreated subjects with viral loads persistently ,50 copies/mL for a median observation time of 28.8 months. HIV-DNA and residual viraemia values and demographic, virological and immunological data were collected for each subject. Results: Undetectable HIV-DNA was found in 16.6% (70/420) of patients and was signicantly correlated with undetectable (,1 copy/mL) plasma viraemia (P 0.0001). Higher CD4 cell count nadir (P,0.001), a lower HIV-RNA viraemia at the start of treatment (P 0.0016) and nevirapine use (P,0.001) were correlated with an undetectable value of HIV-RNA. Twenty-six out of 420 patients (6.2%) reached the treatment goal. In multivariate analysis, higher nadir CD4 cell count (OR 3.86, 95% CI 1.47 10.16, P 0.006), the duration of therapy (OR 1.07, 95% CI 1.021.12, P 0.004) and the use of nevirapine (OR 2.59, 95% CI 1.076.28, P 0.034) were independently related to this condition. Conclusions: Only 6.2% of ART-responder patients presented the combination of three laboratory markers that identied them as full responders. These results indicate the high variability of the ART-responding population and lead us to suggest caution in the selection of patients for possible simplication regimens Keywords: proviral HIV-DNA, residual viraemia, ART

Introduction
Combination antiretroviral treatment (ART) of individuals infected with HIV reduces progression to AIDS and the morbidity and mortality associated with advanced infection.1 3 The prognostic power of numerous laboratory markers has been used to monitor disease progression and response to treatment, but no single parameter has been demonstrated to be more efcient than any other in identifying patients presenting a complete viro-immunological response to ART.

Prolonged ART is required to completely suppress viraemia. Lack of treatment adherence and drug toxicity are the most frequent treatment failure-related factors.4,5 A number of approaches to treatment regimen simplication have been explored to improve adherence, reduce the risk of virological failure and long-term toxicity and enhance patient quality of life,6 9 but it remains difcult to determine the best time for treatment simplication and what parameters are especially useful in dening the category of patients who can most benet from simplication schemes without incurring a risk of

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reduced treatment efcacy. Several clinical ART simplication trials have demonstrated that different parameters in immunovirological responders are correlated with failure. Low adherence, low CD4 nadir level, low CD4 count at simplication and reduced time of virological suppression are all correlated with failure of simplication regimens.10 12 Recently, a trial of patients receiving treatment simplied to darunavir/ritonavir monotherapy indicated a higher risk of failure compared with patients maintained on triple-drug regimens. A multivariate analysis indicated that higher HIV-DNA levels and a plasma viraemia level .1 copy/mL were signicantly correlated with simplication treatment failure.13 With the ultimate aim of identifying the characteristics of the subjects with the best ART response, we have arbitrarily chosen a combination of three laboratory parameters for analysis, as follows: ,10 copies of HIV-DNA/106 peripheral blood mononuclear cells (PBMCs), ,1 copy/mL of plasma HIV-RNA and a CD4 cell count of .500 cells/mm3. We also evaluated the correlations of the combination of these three markers in a group of 420 ART-responding patients.

Cell-associated HIV-1-DNA quantication

Blood collected in EDTA was separated into plasma and cells by Ficoll Hypaque density gradient centrifugation. Next, dry pellets made up of 2106 PBMCs were stored at 2208C until used. To quantify the total proviral HIV-DNA copy number in PBMCs, the real-time TaqMan protocol published by Viard et al.14 was adapted for a LightCycler (Roche Molecular Biochemicals, Indianapolis, IN, USA). The cell line 8E5, containing one integrated HIV-DNA copy per cell, was used to build a standard curve of seven dilutions in duplicate (75 000, 37500, 3750, 375, 37.5, 3.75 and 1.87 copies). The PCR sensitivity was 1 copy of HIV-DNA per reaction (1 copy per reaction is approximately 10 copies/106 PBMCs). The HIV-DNA target was hybridized with a TaqMan probe and read on channel F1/F2 of the LightCycler. To verify DNA integrity and exclude false negatives, we utilized LC control DNA Kit reagents (Roche Molecular Biochemicals) to amplify a 110 bp fragment of human b-globin in the same reaction. This second internal control target was hybridized with a uorescence resonance energy transfer (FRET) probe and read on channel F3/F2 of the LightCycler. To verify the accuracy of the real-time PCR result, different HIV-DNA standards (AIDS Research and Reference Reagent Program, DAIDS, NIAID, NIH:PCR Panel 001 from Dr Shirley Kwok and Dr Cindy Christopherson, Roche Molecular Systems) were also quantied.

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Statistical analysis

Patients and methods

Study design
A cross-sectional, open-label, multicentre trial was conducted in a cohort of 420 HIV-infected subjects retrospectively selected from among those who achieved virological suppression within 6 months of receipt of a rstline therapy and who maintained a plasma HIV-RNA level ,50 copies/ mL, without virological failure, until the follow-up evaluation. A change of the ART, when it occurred, was accepted only if it was due to intolerance or pill burden convenience. Enrolled patients were allowed no more than one viral blip (dened as ,400 HIV-RNA copies/mL) after achieving viral suppression, as determined by frequent blood sampling (at least three times per year). A blood sample was collected from each patient enrolled in the study. Blood samples collected in EDTA-containing tubes were separated into plasma and cells by Ficoll Hypaque density gradient centrifugation. Aliquots of plasma and peripheral blood lymphocytes were stored at 2808C until use. All of the patients had a high level (.95%) of self-reported adherence, and written informed consent was obtained from all of the participants. The Ethics Committee of Tor Vergata University Hospital approved the study.

Unadjusted ORs and 95% CIs from univariate logistic regression models were used to summarize the bivariate associations between each of the demographic, clinical and viro-immunological variables and the different antiretroviral regimens in use with ,10 copies/106 HIV-DNA in PBMCs, ,1 copy/mL of plasma HIV-RNA and a CD4 cell count of .500 cells/mm3. Multivariate logistic regression analysis was used to estimate the adjusted OR and 95% CI of the combination of the three parameters (,10 copies/106 HIV-DNA in PBMCs, ,1 copy/mL of plasma HIV-RNA and a CD4 cell count of .500 cells/mm3). The covariates were selected on the basis of literature data and the signicance of univariate and bivariate analyses. Logistic regression analyses were performed to identify potential confounders. Statistical analyses were performed using the SAS statistical package (SAS Institute Inc., Cary, NC, USA).

Results
The general characteristics of the study population are reported in Table 1. A total of 420 HIV-1-infected patients, receiving rstline ART, with HIV-1 viral loads persistently ,50 copies/mL and without a history of virological failure were included. Out of the total study population, 118 (28.1%) patients were female. The median participant age was 42.0 years (IQR 36.4 47 years). Unprotected heterosexual relationships were the most common risk factor, observed in 45.9% of cases. Median nadir CD4 cell count was 232 cells/mm3 (IQR 105356 cells/mm3). Patients had been on ART for a median of 28.8 months (IQR, 16.8 51.6) and were treated with two nucleoside reverse transcriptase inhibitors (NRTIs) plus a protease inhibitor (PI) in 192 (45.7%) cases or plus a non-nucleoside reverse transcriptase inhibitor (NNRTI) in 228 (54.3%) cases. According to a cross-sectional analysis, the median baseline CD4 cell count was 530 cells/mm3 (IQR 386 725 cells/mm3). Overall, 222 (52.8%) patients had .500 CD4 cells/mm3 and were signicantly younger and had higher nadir CD4 cell counts than patients with a CD4 cell count ,500 cells/mm3 (40.9 versus 43 years, P 0.0024 and 316 versus 143 cells/ mm3, P,0.0001, respectively; data not shown). The median

HIV-1-RNA residual plasma viraemia quantication

Plasma obtained from blood samples collected in EDTA was frozen at 2808C within 4 h of collection and kept at this temperature until tested. Residual viraemia was quantied by an ultrasensitive method based on a modied protocol that used the Amplicor HIV-1 Monitor Kit v1.5 (Roche Molecular Systems, Branchburg, NJ, USA), with a limit of detection of 1 copy/mL. Modications included pelleting of the virus from 2 mL of plasma at 200000 g at 48C for 1 h. The HIV-RNA pellet was extracted, adding half of the normal volume of quantication standard, resuspended in 1/8 of standard volume diluents and assayed by reverse transcription and PCR amplication with a standard protocol. PCR and detection steps followed the manufacturers protocol. For the calculation of HIV-RNA copy number the correction factor was adjusted according to the modied volume of internal quantication standard.

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compared with patients with HIV-RNA levels .1 copy/mL (29.1% versus 9.6%, P,0.0001). Twenty-six out of 420 patients (6.2%) presented a combination of undetectable HIV-RNA plasma levels (,1 copy/mL) and cellular HIV-DNA levels (,10 copies/106 PBMCs) and .500 CD4 cells/mm3. These patients were considered to have the best ART response, and the association of the three parameters was dened as the treatment goal. The comparison of these patients with the remainder of the study population is shown in Table 4. The patients reaching the treatment goal had a signicantly higher CD4 cell count nadir (P 0.019), lower plasma HIV-RNA values at the beginning of therapy (P 0.0006) and longer ART periods (P 0.013); moreover, they were more frequently treated with nevirapine (P 0.013) compared with the 394 patients not reaching the treatment goal. Multivariate analyses, adjusted for multiple confounding variables, are presented in Table 5. Reaching the treatment goal (.500 CD4 cells/mm3, ,1 copy HIV-RNA/mL, ,10 copies HIV-DNA/106 PBMCs) was conrmed to be independently related to the nadir CD4 cell count (OR 3.86, 95% CI 1.47 10.16, P 0.006), the duration of treatment (OR 1.07, 95% CI 1.02 1.12, P 0.004) and the receipt of current nevirapine therapy (OR 2.59, 95% CI 1.07 6.28, P 0.034).

Table 1. Demographic and viro-immunological characteristics of 420 patients with HIV-RNA ,50 copies/mL Patients with HIV-RNA ,50 copies/mL (n420) 42 (36.4 47) 118 (28.1) 193 (45.9) 160 (38.1) 70 (16.6) 232 (105 356) 16 (9 22) 530 (386 725) 28 (20.8 35.4) 5.0 (4.38 5.47) 6 (0 30) 186 (49 412) 28.8 (16.8 51.6) 192 (45.7) 228 (54.3) 159 (37.8) 69 (16.4)

Characteristic Age, yearsa Female, n (%) HIV risk factor, n (%) heterosexual relationship homosexual relationship injection drug use Nadir CD4 cells/mm3a Nadir % of CD4 cellsa CD4 cells/mm3a % of CD4 cellsa Pre-HAART HIV-RNA, log10 copies/mLa Ultrasensitive HIV-RNA, copies/mLa Cellular HIV-DNA, copies/106 PBMCsa Duration of HAART, yearsa Current therapy, n (%) PI NNRTI efavirenz nevirapine
a

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Discussion
Median (IQR).

baseline residual viral load was 6.0 copies/mL (IQR ,1 30 copies/mL), and the baseline cell-associated HIV-DNA copy number was 186 copies/106 PBMCs (IQR 49 412 copies/106 PBMCs). The demographic and viro-immunological characteristics of the patients with .10 or ,10 HIV-DNA copies/106 PBMCs are listed in Table 2. Proviral DNA was undetectable (,10 copies/ 106 PBMCs) in 70 patients (16.6%). No signicant differences were found between the two groups of patients, but heterosexual relationships were the most frequent risk factor in the group of patients with an undetectable level of HIV-DNA (P 0.01), and homosexual patients more frequently had a detectable HIV-DNA level (P 0.07). Moreover, the patients with an undetectable HIV-DNA level were on ART for a longer period. A signicant correlation was found between undetectable HIV-DNA levels and residual viraemia (P 0.0001). In particular, a signicantly higher number of patients with undetectable HIV-DNA levels had undetectable HIV plasma viraemia compared with patients with .10 HIV-DNA copies/106 PBMCs (62.8% versus 30.5%, P,0.0001). The demographic and viro-immunological characteristics of the patients with .1 (n269, 64%) or ,1 (n 151, 35.9%) copy/mL of plasma HIV-RNA are listed in Table 3. The patients with undetectable residual viraemia had a higher CD4 cell count nadir (P,0.001) and lower HIV-RNA viraemia at the start of treatment (P 0.0016); moreover, these patients were more frequently treated with NNRTIs (P 0.008), particularly nevirapine (P,0.001), than patients with detectable levels of HIV-RNA. A signicantly higher number of patients presenting with undetectable viraemia had an undetectable HIV-DNA level

In this cross-sectional multicentre study of 420 HIV-1-infected subjects with no history of virological failure who received ART treatment for .2 years, 26 (6.2%) patients presented with a CD4 cell count .500 cells/mm3, undetectable plasma HIV-RNA levels (,1 copy/mL) and undetectable cellular HIV-DNA levels (,10 copies/106 PBMCs). Higher nadir CD4 cell count, the duration of therapy and the use of nevirapine were independently related to having all three characteristics. Several studies with varying results have reported on the prognostic strength of HIV-DNA.15 24 HIV-DNA is a strong predictor of HIV-1 disease progression, and undetectable HIV-DNA levels have been related to low levels of HIV-DNA pre-therapy, duration of effective ART, high CD4 count at the start of therapy or treatment in the acute infection phase.16 24 In our study, 16.6% of the patients receiving effective highly active antiretroviral therapy (HAART) regimens presented undetectable levels of HIV-DNA, and this characteristic was correlated with undetectable HIV-RNA levels and a prolonged ART period. In a longitudinal study of a cohort of HIV-seropositive subjects evaluated before and at denite timepoints after the beginning of treatment, the achievement of HIV-RNA levels ,2.5 copies/ mL was correlated with low cellular HIV-DNA levels and low plasma viraemia at the beginning of therapy and with high CD4 cell count at baseline and follow-up.19 Approximately 36% (151/420) of the subjects in our study cohort had HIV-RNA levels ,1 copy/mL. This nding was signicantly related to a higher CD4 cell count nadir and, according to the aforementioned longitudinal study, lower HIV-RNA viraemia at the start of treatment and NNRTI use, particularly nevirapine. The superior virological efcacy of nevirapine compared with efavirenz could be related to the greater tissue penetration of the drug. One might suppose that some of the virions remaining measurable

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Table 2. Demographic and viro-immunological characteristics of patients with .10 or ,10 HIV-DNA copies/106 PBMCs Patients with ,10 HIV-DNA copies/106 PBMCs (n70) 42 (35.254.5) 46 (65.7) 41 (58.5) 20 (28.5) 9 (12.8) 270 (70 360) 513 (338 690) 4.73 (2.995.33) 0 (0 5) 44 (62.8) 36 (24 60) 34 36 21 15 (48.6) (51.4) (30) (21.4) Patients with .10 HIV-DNA copies/106 PBMCs (n350) 41 (36.746) 256 (73.1) 149 (42.5) 140 (40) 61 (17.4) 222 (110350) 523 (390709) 4.85 (3.945.32) 236 (102439) 6 (022.9) 107 (30.5) 28 (17 48) 158 (45.1) 192 (54.9) 138 (39.4) 54 (15.4)

Characteristic Age, yearsa Male, n (%) HIV risk factor, n (%) heterosexual relationship homosexual relationship injection drug use Nadir CD4 cells/mm3a CD4 cells/mm3a Pre-ART HIV-RNA, log10 copies/mLa Cellular HIV-DNA, copies/106 PBMCsa Ultrasensitive HIV-RNA, copies/mLa Patients with HIV-RNA ,1 copy/mL, n (%) Duration of ART, monthsa Current therapy, n (%) PI NNRTI efavirenz nevirapine
a

OR (95% CI) 1.01 (0.99 1.04) 0.70 (0.40 1.21) 1.90 (1.13 3.21) 0.60 (0.34 1.05) 0.69 (0.33 1.48) 1.22 (0.73 2.05) 1.00 (0.99 100) 0.88 (0.72 106) 0.94 (0.91 0.97) 3.84 (2.25 6.56) 1.01 (0.98 1.04) 1.14 (0.68 19) 0.87 (0.52 1.45) 0.65 (0.37 1.14) 1.69 (0.78 2.83)

P value 0.09 0.20 0.01 0.07 0.35 0.43 0.55 0.19 0.0001 ,0.0001 0.30 0.59 0.59 0.13 0.21

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Median (IQR).

Table 3. Demographic and viro-immunological characteristics of patients with HIV-RNA .1 or ,1 copy/mL Patients with HIV-RNA ,1 copy/mL (n151) 41 (35 47) 106 (70.1) 69 (45.6) 59 (39.0) 23 (15.2) 300 (167 412) 570 (413 790) 4.39 (2.90 5.00) / 68 (10 305) 44 (29.1) 28 (17 52) 56 (37.1) 95 (62.9) 54 (35.8) 41 (27.2) Patients with HIV-RNA .1 copy/mL (n 269) 42 (37 47) 196 (72.8) 121 (44.9) 101 (37.5) 47 (17.4) 198 (87 298) 490 (364680) 5.0 (4.215.41) 9 (5.631) 222 (82 426) 26 (9.6) 28 (16 48) 136 133 105 28 (50.6) (49.4) (39) (10.4)

Characteristic Age, yearsa Male, n (%) HIV risk factor, n (%) heterosexual relationship homosexual relationship injection drug use Nadir CD4 cells/mm3a CD4 cells/mm3a Pre-ART HIV-RNA, log10 copies/mLa Ultrasensitive HIV-RNA, copies/mLa Cellular HIV-DNA, copies/106 PBMCsa Patients with HIV-DNA ,10 copies/106 PBMCs, n (%) Duration of ART, monthsa Current therapy, n (%) PI NNRTI efavirenz nevirapine
a

OR (95% CI) 0.98 (0.971.00) 0.87 (0.561.36) 1.02 (0.691.53) 1.06 (0.701.60) 0.84 (0.491.46) 2.27 (1.503.41) 1.001 (1.000 1.002) 0.76 (0.650.90) / 1.000 (0.991.00) 3.84 (2.256.56) 1.00 (0.981.03) 0.57 (0.380.86) 1.73 (1.152.60) 0.87 (0.571.31) 3.20 (1.885.45)

P value 0.24 0.56 0.88 0.75 0.55 ,0.001 0.003 0.0016 / 0.12 ,0.0001 0.52 0.008 0.008 0.50 ,0.001

Median (IQR).

by ultrasensitive assay are produced in certain body compartments where nevirapine is able to penetrate and exert a stronger inhibitory effect compared with either efavirenz or protease inhibitors. Interestingly, a signicant majority of patients with an

HIV-RNA level ,1 copy/mL also had undetectable HIV-DNA levels. In previous cross-sectional studies of cohorts of virologically suppressed ART recipients, nevirapine use was the only independent factor associated with virological suppression.25,26

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Patients not achieving treatment goal (n394) 41 (36.3 47) 285 (72.3) 176 (44.6) 153 (38.8) 65 (16.4) 220 (98 348) 498 (372 690) 4.90 (4.03 5.35) 5.9 (0 21.9) 188 (62 412) 28 (17 48) 179 (45.4) 215 (54.6) 155 (39.3) 60 (15.2) OR (95% CI) 1.01 (0.981.05) 0.72 (0.311.66) 1.44 (0.653.20) 0.58 (0.231.41) 1.20 (0.433.31) 2.88 (1.187.01) 0.62 (0.470.81) 0.94 (0.900.98) 1.20 (0.542.65) 0.83 (0.371.84) 0.28 (0.090.82) 2.94 (1.256.91) P value 0.40 0.44 0.36 0.23 0.71 0.019 0.0006 0.013 0.65 0.65 0.021 0.013

Table 4. Demographic and viro-immunological characteristics of patients achieving or not achieving the treatment goal (.500 CD4 cells/mm3, ,1 copy HIV-RNA/mL, ,10 copies HIV-DNA/106 PBMCs) Patients achieving treatment goal (n26) 42.5 (38.253.7) 17 (65.3) 14 (53.8) 7 (26.9) 5 (19.2) 329 (224 460) 665 (575 858) 3.00 (1.4 4.8) 44 (24 76) 13 (50) 13 (50) 4 (15.4) 9 (34.6)

Characteristic Age, yearsa Male, n (%) HIV risk factor, n (%) heterosexual relationship homosexual relationship injection drug use Nadir CD4 cells/mm3a CD4 cells/mm3a Pre-ART HIV-RNA, log10 copies/mLa Ultrasensitive HIV-RNA, copies/mLa Cellular HIV-DNA, copies/106 PBMCsa Duration of ART, monthsa Current therapy, n (%) PI NNRTI efavirenz nevirapine
a

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Median (IQR).

Table 5. Multivariate analysis of patients reaching the treatment goal (.500 CD4 cells/mm3, ,1 copy HIV-RNA/mL, ,10 copies HIV-DNA/106 PBMCs) Adjusted OR Age, years Duration of ART, months Nadir CD4 cells/mm3 Current therapy with nevirapine 1.01 1.07 3.86 2.59 95% CI 0.98 1.05 1.02 1.12 1.47 10.16 1.07 6.28 P value 0.34 0.004 0.006 0.034

A study examining the correlation between cellular HIV-DNA and HIV-RNA levels below laboratory testing thresholds27 demonstrated that there is a strict correlation between sustained undetectable HIV-RNA levels and a low number of cells carrying HIV-DNA. Similar data were obtained in another study,19 which substantially conrms the predictive capacity of HIV-DNA load for the long-term virological success of ART. On the basis of laboratory parameters usually used in the monitoring of antiretroviral therapy, we selected a subset of the studied population with the best viro-immunological characteristics of response to ART. These characteristics were arbitrarily chosen to be CD4 cell count .500 cells/mm3, HIVDNA ,10 copies/106 PBMCs and HIV-RNA ,1 copy/mL. Only 26 patients out of 420 (6.2%) possessed all three characteristics. A multivariate analysis indicated that possessing all three characteristics was signicantly related to nevirapine use, duration of treatment and higher CD4 nadir cell count. The use of NNRTIs, particularly nevirapine, has been associated with a better virological performance in several studies.22,25,27,28

This relationship was originally described in studies that compared NNRTI regimens with unboosted PI regimens, such as the FIRST, ACTG 384 and INITIO clinical trials.29 31 In the ACTG 5142 clinical trial comparing efavirenz- versus lopinavir-including regimens in naive patients, a signicantly better virological outcome was observed at week 96 in patients treated with NNRTIs (P 0.003).32 In a previous publication,23 it was demonstrated that the lowest level of residual viraemia is independently related to the use of NNRTIs, and this result was conrmed by subsequently published studies22,25 and in a recent publication on 1214 patients in which subjects treated with an NNRTI-based ART had a higher chance of maintaining viraemia ,3 copies/ mL.28 Certain authors have attributed this nding to the better penetration of nevirapine in extravascular compartments, such as the CNS, genital tract and breast milk. The good penetration of nevirapine in these areas likely contributes to the local control of viral replication in sanctuaries that are typically spared from the action of other antiretroviral drugs. There are no data on the inuence of NNRTI use on HIV-DNA levels, but an association was proposed between the use of PIs and lower levels of HIV-DNA.22 The timing of the ART initiation and ART duration are related to the control of HIV replication. Our results demonstrate a signicant correlation between combined high CD4 count at nadir and a prolonged effective ART period with undetectable HIV-DNA and HIV-RNA levels, suggesting more efcient virological control following earlier therapy. A correlation between undetectable HIV-DNA levels and the early initiation of ART has been demonstrated in several studies. The correlation between undetectable HIV-DNA levels and ART treatment administered during the primary phase of infection was demonstrated in a longitudinal study on a cohort of HIV-infected people evaluated

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before the initiation of ART and after the virological response to the treatment.19,33 Moreover, in a previous study we demonstrated HIV-DNA reduction to undetectable levels in patients who began treatment with a CD4 cell count .500 cells/mm3.20 The rate of HIV-DNA load decline has been clearly associated with the long-term success of HAART,34 36 and in a group of virological responder patients undetectable HIV-DNA values were independently associated with a prolonged ART duration.21 The main limitation of the study is the cross-sectional design and the lack of HIV-DNA levels prior to therapy because we cannot exclude that they could have inuenced the results obtained. In conclusion, only a few subjects from our cohort of responder ART subjects presented the combination of three laboratory markers that identied them as full responders. These results allowed us to consider the ART virological population as a heterogeneous group of patients in which only a limited number of subjects completely achieved the parameters associated with immunological and virological success. Full response was strictly related to specic characteristics: therapy duration, high CD4 cell count nadir and nevirapine use. The combination of the three aforementioned laboratory parameters may be useful in the evaluation of response to treatment as a guarantee of good control of the infection and should be carefully considered, especially in the selection of patients for possible simplication regimens.

7 Parienti JJ, Bangsberg D, Verdon R et al. Better adherence with once-daily antiretroviral regimens: a meta-analysis. Clin Infect Dis 2009; 48: 4848. 8 Willig JH, Abroms S, Westfall AO et al. Increased regimen durability in the era of once-daily xed-dose combination antiretroviral therapy. AIDS 2008; 22: 1951 60. 9 Santos JR, Molto J, Llibre JM et al. Antiretroviral simplication with darunavir/ritonavir monotherapy in routine clinical practice: safety, effectiveness, and impact on lipid prole. PLoS One 2012; 7: e37442. 10 Arribas JR, Pulido F, Delgado R et al. Lopinavir/ritonavir as single-drug therapy for maintenance of HIV-1 viral suppression: 48-week results of a randomized, controlled, open-label, proof-of-concept pilot clinical trial (OK Study). J Acquir Immune Dec Syndr 2005; 40: 2807. 11 Campo RE, Da Silva BA, Cotte L et al. Predictors of loss of virologic response in subjects who simplied to lopinavir/ritonavir monotherapy from lopinavir/ritonavir plus zidovudine/lamivudine. AIDS Res Hum Retroviruses 2009; 25: 26975.

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12 Pulido F, Perez-Valero I, Delgado R et al. Risk factors for loss of virological suppression in patients receiving lopinavir/ritonavir monotherapy for maintenance of HIV suppression. Antivir Ther 2009; 14: 195201. 13 Lambert-Niclot S, Flandre P, Valantin MA et al. Factors associated with virological failure in HIV-1-infected patients receiving darunavir/ritonavir monotherapy. J Infect Dis 2011; 204: 1211 6. 14 Viard J-P, Burgard M, Hubert J-B et al. Impact of 5 years of maximally successful highly active antiretroviral therapy on CD4 cell count and HIV-1 DNA level. AIDS 2004; 18: 45 9. 15 Tsiara CG, Nikolopoulos GK, Bagos PG et al. Impact of HIV type 1 DNA levels on spontaneous disease progression: a meta-analysis. AIDS Res Hum Retroviruses 2012; 28: 366 73. ` 16 Avettand-Fenoel V, Boufassa F, Galimand J et al. for the ANRS SEROCO Cohort Study Group. HIV-1 DNA for the measurement of the HIV reservoir is predictive of disease progression in seroconverters whatever the mode of result expression is. J Clin Virol 2008; 42: 399404. 17 Hatzakis AE, Touloumi G, Pantazis N et al. Cellular HIV-1 DNA load predicts HIV-RNA rebound and the outcome of highly active antiretroviral therapy. AIDS 2004; 18: 2261 7. 18 Minga AK, Anglaret X, d Aquin Toni T et al. HIV-1 DNA in peripheral blood mononuclear cells is strongly associated with HIV-1 disease progression in recently infected West African adults. J Acquir Immune Dec Syndr 2008; 48: 350 4. 19 Parisi SG, Andreis S, Mengoli C et al. Baseline cellular HIV DNA load predicts HIV DNA decline and residual HIV plasma levels during effective antiretroviral therapy. J Clin Microbiol 2012; 50: 25863. 20 Andreoni M, Parisi SG, Sarmati L et al. Cellular proviral HIV-DNA decline and viral isolation in nave subjects with ,5000 copies/mL of HIV-RNA and .500106/L CD4 cells treated with highly active antiretroviral therapy. AIDS 2000; 14: 23 9. 21 Sarmati L, Parisi SG, Nicastri E et al. Association between cellular human immunodeciency virus DNA level and immunological parameters in patients with undetectable plasma viremia level during highly active antiretroviral therapy. J Clin Microbiol 2005; 43: 6183 5. 22 Nicastri E, Palmisano L, Sarmati L et al. HIV-1 residual viremia and proviral DNA in patients with suppressed plasma viral load (,400 HIV-RNA cp/mL) during different antiretroviral regimens. Curr HIV Res 2008; 6: 2616. 23 Sarmati L, Andreoni C, Nicastri E et al. Prognostic factors of long-term CD4+ count-guided interruption of antiretroviral treatment. J Med Virol 2009; 81: 4817.

Funding
This work was supported by European AIDS Treatment Network (NEAT) contract LSHP-CT-2006-037570. A Network of Excellence EC project in the FP 6 programme under integrating and strengthening the European Research Area.

Transparency declarations
None to declare.

References
1 Zolopa A, Andersen J, Powderly W et al. Early antiretroviral therapy reduces AIDS progression/death in individuals with acute opportunistic infections: a multicenter randomized strategy trial. PLoS One 2009; 4: e5575. 2 Mocroft A, Vella S, Beneld TL et al. Changing patterns of mortality across Europe in patients infected with HIV-1. EuroSIDA Study Group. Lancet 1998; 352: 1725 30. 3 Hogg RS, Yip B, Chan KJ et al. Rates of disease progression by baseline CD4 cell count and viral load after initiating triple-drug therapy. JAMA 2001; 286: 2568 77. 4 Gallant JE, Staszewski S, Pozniak AL et al. Efcacy and safety of tenofovir DF vs stavudine in combination therapy in antiretroviral-nave patients: a 3-year randomized trial. JAMA 2004; 292: 191201. 5 Gulick RM, Meibohm A, Havlir D et al. Six-year follow-up of HIV-1-infected adults in a clinical trial of antiretroviral therapy with indinavir, zidovudine, and lamivudine. AIDS 2003; 17: 2345 9. 6 McKinnon JE, Mellors JW, Swindells S. Simplication strategies to reduce antiretroviral drug exposure: progress and prospects. Antivir Ther 2009; 14: 1 12.

6 of 7

Undetectable HIV-RNA and HIV-DNA levels during ART

JAC
30 Robbins GK, De Gruttola V, Shafer RW et al. AIDS Clinical Trials Group 384 Team. Comparison of sequential three-drug regimens as initial therapy for HIV-1 infection. N Engl J Med 2003; 349: 2293 303. 31 INITIO Trial International Co-ordinating Committee, Yeni P, Cooper DA et al. Virological and immunological outcomes at 3 years after starting antiretroviral therapy with regimens containing non-nucleoside reverse transcriptase inhibitor, protease inhibitor, or both in INITIO: open-label randomised trial. Lancet 2006; 368: 287 98. 32 Riddler SA, Haubrich R, DiRienzo AG et al. AIDS Clinical Trials Group Study A5142 Team. Class-sparing regimens for initial treatment of HIV-1 infection. N Engl J Med 2008; 358: 2095 106. 33 Ananworanich J, Schuetz A, Vandergeeten C et al. RV254/SEARCH 010 Study Group. Impact of multi-targeted antiretroviral treatment on gut T cell depletion and HIV reservoir seeding during acute HIV infection. PLoS One 2012; 7: e33948. 34 Ngo-Giang-Huong N, Deveau C, Da Silva I et al. French PRIMO Cohort Study Group. Proviral HIV-1 DNA in subjects followed since primary HIV-1 infection who suppress plasma viral load after one year of highly active antiretroviral therapy. AIDS 2001; 15: 665 73. 35 Pellegrin I, Caumont A, Garrigue I et al. Predictive value of provirus load and DNA human immunodeciency virus genotype for successful abacavir-based simplied therapy. J Infect Dis 2003; 187: 38 46. 36 Ibanez A, Puig T, Elias J et al. Quantication of integrated and total HIV-1 DNA after long-term highly active antiretroviral therapy in HIV-1-infected patients. AIDS 1999; 13: 1045 9.

24 Sarmati L, Parisi SG, Nicastri E et al. Cellular HIV-1 DNA quantitation in patients during simplication therapy with protease inhibitor-sparing regimens. J Med Virol 2007; 79: 8806. 25 Bonora S, Nicastri E, Calcagno A et al. Ultrasensitive assessment of residual HIV viraemia in HAART-treated patients with persistently undetectable plasma HIV-RNA: a cross-sectional evaluation. J Med Virol 2009; 81: 4005. 26 Ham-Boukobza S, Morand-Joubert L, Flandre P et al. Higher efcacy of nevirapine than efavirenz to achieve HIV-1 plasma viral load below 1 copy/mL. AIDS 2011; 25: 3414. 27 Chun TW, Murray D, Justement JS et al. Relationship between residual plasma viremia and the size of HIV proviral DNA reservoirs in infected individuals receiving effective antiretroviral therapy. J Infect Dis 2011; 204: 1358. 28 Maggiolo F, Callegaro A, Cologni G et al. Ultrasensitive assessment of residual low-level HIV viremia in HAART-treated patients and risk of virological failure. J Acquir Immune Dec Syndr 2012; 60: 47382. 29 MacArthur RD, Novak RM, Peng G et al. CPCRA 058 Study Team; Terry Beirn Community Programs for Clinical Research on AIDS (CPCRA). A comparison of three highly active antiretroviral treatment strategies consisting of non-nucleoside reverse transcriptase inhibitors, protease inhibitors, or both in the presence of nucleoside reverse transcriptase inhibitors as initial therapy (CPCRA 058 FIRST Study): a long-term randomised trial. Lancet 2006; 368: 2125 35.

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