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Baseline Cellular HIV DNA Load Predicts HIV DNA Decline and Residual 1
HIV Plasma Levels During Effective Antiretroviral Therapy. 2
3
Parisi Saverio Giuseppe *1, Andreis Samantha1, Mengoli Carlo1, Scaggiante Renzo2, Ferretto 4
Roberto3, Manfrin Vinicio4, Cruciani Mario5, Giobbia Mario6, Boldrin Caterina1, Basso 5
Monica1, Andreoni Massimo7, Palù Giorgio1, Sarmati Loredana7 6
7
1Department of Histology, Microbiology and Medical Biotechnology, University of Padova, 8
Padova, Italy 9
2Infectious Diseases Unit, Padova Hospital, Padova, Italy 10
3Infectious Diseases Unit, Schio Hospital, Schio, Italy 11
4Infectious Diseases Unit, Vicenza Hospital, Vicenza, Italy 12
5Center of Preventive Medicine & HIV Outpatient Clinic, Verona, Italy 13
6Infectious Diseases Unit, Treviso Hospital, Treviso, Italy 14
7Department of Public Health, University of Rome Tor Vergata, Rome, Italy 15
16
Running title: Cellular HIV DNA and Residual Viremia 17
18
*Corresponding author. Mailing address: 19
Department of Histology, Microbiology and Medical Biotechnology, Padova University, Via 20
Gabelli 63, 35100 Padova, Italy 21
Phone: +39-049-8272344 22
Fax: +39-049-8272355. 23
E-mail: [email protected] 24
25
26
Copyright © 2011, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.J. Clin. Microbiol. doi:10.1128/JCM.06022-11 JCM Accepts, published online ahead of print on 30 November 2011
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Abstract 27
28
Cellular human immunodeficiency virus type 1 (HIV-1) DNA may be considered a marker of 29
disease progression with a significant predictive power: published data on its correlation with 30
plasma HIV RNA and CD4 count, in acute and chronic patients are not conclusive. We 31
evaluated a cohort of 180 patients naïve for antiretroviral therapy before the beginning of 32
treatment and after a virological response in order to define the indicators correlated with HIV 33
DNA load decrease till undetectability. The following variables were evaluated as continuous: 34
age, CD4 cell count and HIV DNA log10 at baseline and follow up, baseline HIV RNA log10. 35
Being in primary HIV infection at start of therapy, HIV RNA at follow up under 2.5 36
copies/ml, origin, gender, transmission risk were evaluated as binary. The decline of HIV 37
DNA values during an effective therapy was directly related to baseline HIV DNA and HIV 38
RNA values at baseline, to the increase in the number of CD4 cells and the achievement of a 39
HIV RNA load <2.5 copies/ml. Undetectable HIV DNA cellular load was achieved by 21.6% 40
of patients at the follow-up time point and significantly correlated with lower baseline cellular 41
HIV DNA values and being in the primary stage of infection when therapy started. In 42
conclusion, early treatment facilitated the achievement of undetectable levels of plasma 43
viraemia and cellular HIV DNA and a better recovery of CD4 lymphocytes. HIV DNA level 44
before and during highly active antiretroviral therapy may be used as a new tool for 45
monitoring treatment efficacy 46
47
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Introduction 48
49
Cellular HIV DNA load directly correlates with the amount of HIV latently infected cells that 50
constitute the viral reservoir and is considered an independent marker of disease progression 51
(16) with a strong predictive power in acute and chronic patients (12, 25). Therefore, cellular 52
HIV DNA load would be also considered a potential indicator for the initiation of highly 53
active antiretroviral therapy (HAART). The HIV DNA load predicts the long-term success of 54
HAART in naïve patients and is able to anticipate virological failure in treated patients. 55
Recently (18), a prospective multicentre study assessed the predictive value of peripheral 56
blood mononuclear cells (PBMCs) HIV-1 DNA on virological and immunological outcomes 57
in a cohort of 148 patients who were treated from 1998 with a first line protease inhibitor (PI) 58
containing regimen and demonstrated that a higher baseline HIV-1 DNA level was associated 59
with an increased risk of virological failure after one year of HAART. This phenomenon was 60
conserved in the long-term (7 years) in the univariate analysis but was no longer significant in 61
the multivariate analysis. No effects of HIV-1 DNA on the immunological outcome were 62
observed in the short-term or in the long-term. A small follow-up study of 51 patients 63
indicated that the HIV DNA load was the only predictive parameter of virological rebound in 64
the group of non-responders (15). Furthermore, in patients with advanced therapeutic failure, 65
a higher HIV DNA load may be associated with lower increases in CD4 count. Therefore, 66
HIV DNA levels have been recently suggested as a useful tool for the case management of 67
patients in the late stages of disease (3). 68
HAART-mediated reductions in HIV DNA levels were studied (29) in a 5-year follow-up of 69
25 patients who were treated with HAART. The study showed that the largest HIV DNA 70
decrease was evident during the first year of therapy followed by a milder decrease during the 71
2nd and 3rd years without any further diminution, suggesting that any additional benefit of 72
treatment in terms of reduction of the viral reservoir was unlikely. The evolution of HIV DNA 73
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was not different for patients with baseline CD4 cell counts that were above or below the 74
median value (127 cells/mm3). 75
In 236 patients receiving successful therapy for more than 3 years (7), a univariate analysis 76
showed that HIV-1 DNA levels did not correlate with therapy duration, time spent with 77
undetectable HIV-1 RNA or the occurrence of a viral blip, defined as less than 1000 HIV 78
RNA copies/ml. Plasma HIV-1 RNA zenith and CD4 cell count nadir remained predictive of 79
HIV-1 DNA levels in the multivariate model. 80
In clinical practice, a significant number of patients reached undetectable HIV DNA levels 81
during HAART. 82
Given the conflicting reports between the correlation among HIV DNA levels and the 83
virological and immunological responses to treatment, we evaluated a cohort of 180 patients 84
who were studied before the beginning of treatment and after a virological response to 85
HAART to elucidate the indicators that correlated with decreased or undetectable HIV DNA 86
load. 87
88
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Methods 89
90
A cohort of 180 HIV-infected subjects, who were naïve for antiretroviral therapy, was 91
enrolled in five Infectious Diseases Units in the Veneto region in Italy. Patients were 92
retrospectively selected among those who achieved virological suppression with the first-line 93
therapy within 6 months and maintained plasma HIV RNA levels <50 copies/ml without 94
virological failures until the follow-up time point evaluation. A blood sample was collected 95
before the beginning of therapy (T0) and at the time of follow-up (T1), which ranged from 6 96
months to 6 years after achieving the virological suppression. 97
In the event of a change of the HAART, it was accepted if this change was due to intolerance 98
or pill burden convenience. Patients who interrupted or needed treatment modifications for 99
failure were excluded from the study. Patients who had more than one viral blip per year after 100
achieving viral suppression, which was determined by frequent blood sampling (at least 4 101
times per year), were not considered useful for the aim of the study and were not included. 102
All of the enrolled subjects gave informed consent to all procedures and the use of their 103
blinded data for a scientific evaluation and publication. The local Government, which was 104
represented by the Veneto Regional Health Authority, approved the study and provided 105
funding. This study was conducted in accordance with the Helsinki Declaration and the local 106
legislation. 107
A primary HIV infection was defined by the presence of the following: (i) a negative or 108
indeterminate HIV antibody enzyme linked immunosorbent assay associated with HIV RNA 109
positive plasma or (ii) an initially negative test for HIV antibodies followed by positive 110
serology within 18 months. 111
HIV genotypic analyses to detect subtypes were performed as previously described (24). 112
Blood samples collected in EDTA-containing tubes were separated into plasma and cells 113
using Ficoll-Paque Plus density gradient centrifugation. Aliquots of plasma and dried pellets 114
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of 2 x 106 PBMCs were stored at -80°C until use. Baseline and follow-up blood samples in 115
EDTA were submitted to the Laboratory of Virology at the University of Padova and stored 116
within 6 hours from collection until analysis. 117
118
Cellular HIV DNA quantitation 119
The extraction and purification of DNA from cells was performed using the QIAmp Blood kit 120
(Qiagen, Inc., Chatsworth, CA). The real-time TaqMan protocol that has been published by 121
Viard and colleagues (29) was used to quantify the cellular HIV DNA copy number in 122
PBMCs. The cell line 8E5, which contains one copy of integrated HIV DNA in each cell, was 123
used to build a standard curve, with a sensitivity of 5 copies/million PBMCs (21). 124
Duplicate analysis within the same experiment was conducted for both HIV and beta-globin 125
evaluation, and a mean of these values was calculated. When delta values were more than 126
10% the sample was analysed again. Baseline and follow up samples of a patient were 127
always evaluated in the same experiment; this procedure was possible because only complete 128
sample sets (T0 and T1) of the patients, retrospectively selected, were considered. A sample 129
previously evaluated was regularly added as internal control. 130
131
Quantification of plasma residual viraemia 132
Plasma samples were obtained from the blood collected and frozen at -80°C within 6 hours 133
from collection and kept at this temperature until tested. Residual viraemia was quantified 134
using an ultra-ultrasensitive method based on a modified Amplicor HIV-1 Monitor test 135
version 1.5 (Roche Molecular Systems, Branchburg, New Jersey, USA), with a limit of 136
detection of 2.5 copies/ml (21). Modifications included pelleting viruses from 2 ml of plasma 137
at 23.600 g at 4°C for 2 hours, adding half of the normal volume of the quantification 138
standard and resuspending the RNA pellet in 50 μl of diluent. The entire volume of 139
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resuspended RNA was assayed using reverse transcription and PCR amplification. These and 140
subsequent detection steps followed the manufacturer’s protocol. 141
Patients were subdivided at the follow-up time point into 4 groups: 1) subjects with 142
undetectable plasma viraemia (PLV) at the time of evaluation, defined as Ultra Low (UL, 143
<2.5 HIV RNA copies/ml); 2) with low level plasma viraemia (LL, 2.5-20 copies/ml); 3) with 144
High Level plasma viremia (HL, 20-50 copies/ml) and 4) with viral blips (VB, >50-1000 145
copies/ml). Patients with PLV over 1000 copies/ml at the follow-up time point were not 146
included. 147
148
Statistical analysis 149
The following variables were analyzed: age, CD4 cell count at baseline and follow up, HIV 150
DNA log10 copies/106 PBMCs at baseline and follow up, HIV RNA log10(copies) /ml at were 151
evaluated as continuous. HIV RNA at follow up under the lowest threshold level (under 2.5 152
copies/ml), primary HIV infection, stranger origin, male gender, homosexual risk, injection 153
drug addict, were evaluated as binary. 154
All variables were submitted to pairwise correlation analysis, in order to outline the 155
underlying link pattern in the evaluated population. The correlation was established by linear 156
regression analysis when at least one variable was continuous. When both paired variables 157
were categorical (binary), the chi-square test was used. 158
Time to event procedures were employed as well. In this context, Kaplan Meier (KM) non 159
parametric approach (with the logrank test) and the semiparametric proportional hazards Cox 160
regression (Cox) were used. The latter was performed aiming at a multivariate scope. 161
A time to event (survival) analysis was performed by using the lowering of follow up HIV 162
DNA load to the level of <5 copies/106 PBMCs as the relevant event. Therefore, the defined 163
event was a favorable outcome, and the term “hazard” or “risk” meant the probability of this 164
outcome. 165
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The effect of the relevant explanatory variables was investigated adopting the Cox semi 166
parametric cumulative hazards regression method. Initially, a bivariate analysis was 167
performed. Thereafter, a multivariate approach was exploited. 168
169
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Results 170
171
The median age of the 180 selected patients was 41 years (range, 19 to 79 years). A total of 172
140 subjects were male (77.8%), and 63/180 subjects (35%) were men who reported having 173
sex with men as a risk factor. A total of 84 patients (46.7%) were heterosexual, 17 patients 174
(9%) were intravenous drug users, and 16 patients had undetermined risk factors. Among 180 175
subjects, 138 subjects (76.7%) were Italians. 176
At the beginning of HAART, the median CD4 count was 250 cells/mm3 (range, 0 to 1090 177
cells/mm3) and the median percentage was 15% (range, 0 to 42 %). The median HIV RNA 178
level was 5 log copies/ml (range, 1.91 to 6.97 log10 copies/ml), and the median HIV DNA 179
level was 3.26 log10 copies/106 PBMCs (range 1.34 to 4.84 log10 copies/106 PBMCs). 180
In 48 subjects (26.6%), a non-B subtype strain was detected. Patients with the non-B subtype 181
were more likely to be younger (P=0.0007), strangers (P<0.0001), females (P<0.0001), and 182
with lower baseline plasma HIV RNA levels (P=0.0075). A total of 21 patients (11.6%) were 183
enrolled and started therapy during the acute infection period. 184
185
186
Trend of HIV DNA and correlation with other parameters 187
188
A reduction of HIV DNA values between T0 and T1 was present in all studied patients. 189
To assess the relationship between the trend of HIV DNA load (from T0 to T1) and the other 190
parameters, a pairwise correlation analysis was performed. The log10 values of cellular HIV 191
DNA at T1 positively correlated with the baseline values of cellular HIV DNA and plasma 192
HIV RNA (P<0.0001 and P=0.0257, respectively), with achieving an undetectable value of 193
plasma HIV RNA (<2.5 cps/ml) at T1, and the condition of primary HIV infection at the start 194
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of therapy (P<0.0001 for both) and negatively correlated with the baseline values of CD4 195
count (P=0.0236). 196
The correlation between the changing values of cellular HIV DNA and CD4 cell counts over 197
time was investigated using a multilevel, mixed effects multivariate regression analysis 198
(Figure 1). The dependent variable was log10 HIV DNA. After a preliminary inclusion of 199
several explanatory covariates, including CD4 cells, HIV RNA (<2.5 copies/ml at T1), being 200
in primary HIV infection at start of therapy, the non-B HIV subtype, age, nationality other 201
than Italian, intravenous drug users, men having sex with men, only the CD4 cell count and 202
HIV RNA levels (<2.5 copies/ml at T1) along with time were retained as significant. Time 203
was also included in the random effects section. The observations were grouped by patients. 204
The figure 1 reports the log10 DNA as a function of CD4, at time points T0 and T1, along with 205
linear predictions. 206
Examining the coefficients, we noted that if the CD4 cell count increased by 1, HIV DNA 207
was multiplied for 0.998 (i.e., 10-0.0017 = 0.998). 208
If the undetectable HIV RNA level at T1 changes from 0 (partial suppression) to 1 (full 209
suppression), DNA is to be multiplied for 10-0.3273 = 0.471. If time increases of one year, DNA 210
is to be multiplied for 10-0.4539 = 0.352. For example,with 400 CD4 cells, HIV DNA is 477 211
copies/ml in a patient with partial plasma viral RNA suppression at T1 (time of follow-up)= 1 212
year .Then, if HIV RNA level at T1 turns from partial to full suppression of plasma viral 213
RNA, DNA changes to 224 copies. In general, HIV DNA is calculated as follows: 2652 x 214
0.998CD4x 0.471RNA-T1x 0.352time, where 103.4237 (=10intercept) = 2652. 215
Investigating the possible role of several covariates (CD4 cell count change, age, gender, 216
nationality other than Italian, men who have sex with men or intravenous drug users risk 217
factors, achieving <2.5 copies/ml of plasma HIV RNA, being in primary infection at the start 218
of therapy and non-B sub-type) for predicting a reduction of the log10 value of cellular HIV 219
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DNA load at T1, a final model showing a significant correlation was obtained with two 220
explanatory covariates that independently exerted a significant effect: the CD4 cell count T0-221
T1 increase (P=0.004, CI 95% 0.001-0.000) and achieving <2.5 copies/ml of plasma HIV 222
RNA (P = 0.022, CI 95% 0.609-0.048). 223
224
225
Undetectable HIV DNA load (<5 copies/106 PBMCs) at T1 and correlation with other 226
parameters. 227
228
To assess whether some parameters (CD4 cell count, baseline log10 DNA and log10 229
RNA, suppression of plasma viremia under 2.5 genomic copies/ml, primary HIV infection, 230
HIV subtype, ethnicity, gender, HIV transmission risk) might be related to achieve 231
undetectable HIV DNA load at T1, a time to event (survival) analysis was performed using 232
the achievement of cellular HIV DNA at <5 copies/106 PBMCs as the relevant event. Thirty-233
nine patients reached this outcome among the 180 patients who were included in the cohort 234
(21.6%). 235
The effect of the relevant explanatory variables was investigated adopting the Cox 236
semiparametric cumulative hazards regression method. Using the bivariate analysis, the 237
dependent variable was the successful event, which was defined as the cellular HIV DNA 238
load under the threshold of <5 copies/106 PBMCs. The achievement of <5 copies/106 PBMCs 239
in the cellular HIV DNA load correlated with lower baseline log cellular HIV DNA levels 240
(P<0.001), achieving the undetectable levels of HIV RNA (<2.5 copies/ml) at T1 (P=0.003) 241
and being in the clinical condition of primary infection at the start of therapy (P=0.006). No 242
correlation was found between an HIV DNA load of <5 copies/106 PBMCs at T1 and baseline 243
values CD4 cell counts. 244
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This approach failed to obtain a significant result when the explanatory variable was baseline 245
log10(RNA). However, a correlation between the dependent variable and the above predictor 246
was demonstrated by two alternative methods, (a) counting the successful proviral DNA 247
suppression events after stratifying the baseline plasma RNA into three levels, <10.000, 248
10.000-99.999, and ≥100.000, and using the Pearson chi2 test thereafter (7.4397, P = 0.024), 249
(b) by ANOVA method, baseline log10(RNA) versus cellular HIV DNA suppression 250
(P=0.0340). 251
Using a multivariate analysis only log10 baseline cellular HIV DNA value maintained an 252
independent predictive role for the achievement of undetectable HIV DNA load at T1 253
(P<0.001; CI 0.211-0.480). In particular it has been demonstrated that an increase of one log 254
in the cellular HIV DNA load at baseline reduced the probability of the favourable event by 255
approximately one-third. 256
In figure 2, the proportion of patients who were exempt from the relevant event (cellular HIV 257
DNA at T1 reduced to <5 copies) was plotted as a function of time (years). The effects of the 258
baseline log10 HIV DNA values were indicated as 10th, 50th, and 90th percentiles, 259
corresponding to 2.42, 3.26 and 3.79 log10 copies/106 PBMCs, respectively. 260
261
Effect of therapy during primary infection 262
263
The main characteristics of the population were assessed using a pairwise correlation analysis, 264
which showed that being in primary infection at the start of therapy positively correlated with 265
high CD4 counts at baseline (P<0.0001) and at T1 (P=0.0076), achieving <2.5 copies/ml of 266
plasma HIV RNA at T1 (P=0.0003), and low cellular HIV DNA levels at T1 (P<0.0001). 267
Achieving HIV RNA levels that were less than 2.5 copies/ml at T1 correlated with low 268
baseline values of plasma HIV RNA (P=0.0007), low baseline cellular HIV DNA (P=0.0004) 269
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and with low cellular HIV DNA at T1 (P<0.0001) and high CD4 cell counts at T0 270
(P<0.0001). 271
272
Effect of HAART on residual plasma viraemia 273
274
At T1, the subjects were subdivided into four groups according to the plasma viraemia values: 275
1) UL = <2.5 copies/ml; 2) LL = 2.5-20 copies/ml; 3) HL = >20-50 copies/ml and 4) VB = 276
>50-1000 copies/ml. Immuno-virological values of these patients are shown in table 1. 277
The effects of the current treatment, in terms of using a different third drug that was 278
associated with a backbone, on the achievement of HIV RNA < 2.5 copies/ml at T1 were 279
evaluated using a semiparametric analysis. 280
Explanatory treatment variables were: efavirenz (71 patients receiving treatment at T1), 281
nevirapine (21 patients), ritonavir-boosted protease inhibitors (83 patients) and the third 282
nucleoside analogue (Trizivir®, 4 patients). The variable non-nucleoside analogues included 283
efavirenz or nevirapine. 284
Using the bivariate Cox regression method, no explanatory treatment variable was significant. 285
Efavirenz is closest to significance (hazard ratio = 1.55, z = 1.84). 286
287
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Discussion 288
289
In the study cohort, the decline of HIV DNA values during an effective therapy was directly 290
related to HIV DNA and HIV RNA values at the beginning of HAART (P<0.0001 and 291
P=0.0257 respectively), gain in the number of CD4 cells (P=0.02369) and achievement of a 292
HIV RNA load <2.5 copies/ml (P<0.0001). 293
Lower HIV DNA load after a defined duration of HAART was significantly related to higher 294
pre-HAART CD4 cell count and percentage, and prolonged duration of antiretroviral 295
treatment (27, 30, 19, 2). Furthermore, the absence of previous exposure to antiretroviral 296
therapy was a predictive factor of an higher decreased cellular HIV-1 DNA levels (19). 297
Finally, in patients with undetectable levels of plasma viraemia after one year of HAART, the 298
highest reduction of HIV DNA was obtained in subjects who started treatment in the early 299
asymptomatic phase of infection with more than 500 CD4 cells/mm3 (2). 300
In our study, 21.6% of patients achieved a HIV DNA cellular load of less than 5 copies/106 301
PBMCs at the follow-up time point. This favourable outcome correlated with lower baseline 302
cellular HIV DNA values (P<0.001) and being in the primary stage of infection at the start of 303
therapy (P=0.006). One log10 increase in the value of HIV DNA load at baseline reduced the 304
probability of reaching <5 copies /106 PBMCs by approximately one-third at the follow-up 305
time. Moreover, a strong correlation between cellular HIV DNA decreases and the CD4 cell 306
count increases over time was demonstrated. 307
In agreement with another study (9), a high proportion of patients (40.5%) achieved 308
undetectable levels of plasma HIV RNA (<2.5 copies/ml) at the follow-up time point.This 309
virological endpoint was correlated with low cellular HIV DNA levels at baseline (P=0.0004) 310
and follow-up (P<0.0001), low baseline plasma viraemia (P=0.0007), and high CD4 cell 311
count at baseline (P<0.0001) and follow-up (P=0.0329). 312
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In a cross-sectional analysis after 2 years of effective HAART, only 5 out of 84 subjects (6%) 313
had undetectable levels of proviral HIV DNA, and 42 subjects had HIV RNA values < 2.5 314
copies/ml (23). The usage of a non-nucleoside reverse transcriptase inhibitors (NNRTI)-based 315
HAART was the only independent predictor of plasma RNA suppression below the cut-off 316
value. 317
The difference can be explained with the characteristic features of the enrolled patients. 318
Different exclusion criteria for virological failure were used. In addition, in the study by 319
Palmisano only 26% of patients were on PI therapy and less than 2% of these patients using 320
boosted protease inhibitor therapy. 321
The correlation between the favourable HIV RNA and HIV DNA outcomes were described in 322
a cross-sectional study by Chun, who reported the frequency of CD4 T cells carrying HIV 323
proviral DNA. When the data on HIV proviral DNA burden were stratified based on residual 324
plasma viremia, a statistically significant difference was observed between the study subjects 325
with undetectable (0 copies) and detectable (1–49 copies) plasma viraemia (P=0.01). These 326
results indicate that the frequency of CD4 T cells carrying HIV proviral DNA in infected 327
individuals with undetectable plasma viraemia is lower than that of individuals with 328
detectable plasma viraemia. In the current study, the results confirm this observation using 329
total PBMCs and provide new insights regarding the predictivity of baseline virological 330
values from the archive. 331
A previous study suggested (22) that low-level persistent viraemia was derived from at least 332
two cell compartments: one compartment demonstrated viral production decay over time, 333
whereas a second compartment maintained stable viral production for at least 7 years. 334
Hatano (14) observed that plasma viraemia continued to decline during the first 12 months 335
after viraemia was undetectable using conventional methods and remained stable, appearing 336
to achieve a steady-state ‘set-point’ during long-term combination therapy. 337
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This residual viraemia was not reduced by treatment intensification (10, 11, 17). However, 338
these results are under debate (8); it was suggested that residual viraemia does not arise from 339
ongoing cycles of HIV-1 replication and infection of new cells. 340
Latently infected, resting CD4 T cells and/or unidentified viral reservoirs are capable of 341
producing low levels of virions for prolonged periods of time in individuals receiving 342
HAART for extended periods of time (4). Previous studies observed that residual plasma 343
viruses and CD4 T cell-associated viruses were separately compartmentalised (26, 6). 344
Nevertheless, in a recent study, clonal sequences recovered from residual HIV-1 viraemia in 345
patients receiving intensified HAART were identical to replicating viral RNAs that were 346
recovered from circulating resting CD4+ T cells (1). 347
Our data showed that an undetectable level of plasma viraemia, low HIV DNA levels and 348
high CD4 counts may be more probably attained by patients who start HAART while having 349
a low level of cellular HIV DNA. 350
The timing of HAART intervention was suggested to have remarkably different effects on 351
HIV DNA compartment. A total of 21 subjects out of 180 subjects in our population started 352
therapy during the phase of primary infection. A total of 16 subjects had less than 2.5 353
copies/ml of HIV RNA at follow-up (P=0.0003). This very early therapy correlated with 354
lower HIV DNA levels at follow-up (P<0.0001) and with achieving a HIV DNA cellular load 355
of less than 5 copies/106 PBMCs (P=0.006). Using a multivariate analysis, only cellular HIV 356
DNA levels maintained an independent predictive role for the achievement of undetectable 357
cellular HIV DNA in the study population. These data are in agreement with data obtained 358
from patients who were treated in the early, asymptomatic phase of infection (2) and in 359
primary infection (20, 30). Finally, it was reported that patients who were treated before or 360
within 6 months after seroconversion harboured smaller reservoirs of recoverable HIV after 1 361
year of HAART compared to patients who were treated for 3-6 years during chronic infection 362
(28). 363
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More potent effects on residual viraemia were demonstrated with the use of NNRTI than with 364
protease inhibitors (21, 5), and a better performance of nevirapine than efavirenz was 365
suggested (5, 13). In this study, no significant association was demonstrated between HAART 366
composition and undetectable levels of HIV DNA. Efavirenz showed the best performance 367
approaching statistical significance (HR=1.55, z=1,84). It’s important to note that the majority 368
of patients received the first-line treatment, and few patients were treated with nevirapine. 369
A bias need to be discussed. Only peripheral HIV DNA was suitable for the analysis, and a 370
measure of a clinical useful and easily accessible parameter was planned; not differentiate 371
between integrated and non-integrated HIV DNA forms makes it difficult to comment on the 372
potential prognostic value of DNA levels, but we were unable to measure the baseline values 373
of non-integrated virus for lacking of adequate quantity of PBMCs for this analysis (about 6 × 374
107 PBMCs are needed) stored at T0; the follow up values without a baseline reference would 375
be not interpretable. 376
In conclusion, low HIV DNA levels during HAART correlates with CD4 and low level 377
residual viremia; treatment during the early phase of HIV infection facilitated the 378
achievement of undetectable levels of plasma viraemia and cellular HIV DNA as well as a 379
better recovery of CD4 lymphocytes. HIV DNA level evaluation before and during HAART 380
may be used as a new tool for monitoring the efficacy of treatment. 381
382
383
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Acknowledgements 384
This work was supported by the Veneto Regional Health Authority and the main institutions 385
involved, the Department of Histology, Microbiology and Medical Biotechnology, Padova 386
University, and the Padova Hospital (D.G.R 3643/04 and D.G.R. 3499/08 to GP). 387
388
Authors’ contributions 389
PSG designed and coordinated the study, supervised laboratory experiments, collected the 390
data, interpreted the findings and wrote the paper; AS performed laboratory experiments, MC 391
interpret the data and performed statistical analysis, RS helped design the study, managed the 392
patients and collected the samples, FR managed the patients and collected the samples, MV 393
managed the patients and collected the samples, CM managed the patients and collected the 394
samples, GM managed the patients and collected the samples, BC performed laboratory 395
experiments, BM helped interpretation of the findings and writing the paper; AM designed the 396
study, helped interpretation of the findings and writing the paper; PG designed the study, 397
helped interpretation of the findings and writing the paper; SL designed the study, helped 398
interpretation of the findings and writing the paper. 399
All authors read and approved the final manuscript. 400
PSG, SR, FR, MV, CM, GM and PG are members of CAVeAT (Cohort of Amici Venetians 401
for Antiretroviral Treatment). 402
403
Preliminary data from this study were presented as oral communication at 50th ICAAC 404
in Boston, in 2010 (H-1167, session 113). 405
406
407
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Figure 1
5
Linear prediction
3
4
us, l
og10
(DN
A)
2
HIV
pro
viru
rnaend=0, time=0rnaend=0, time=1rnaend=1, time=0rnaend=1, time=1
1
0 200 400 600 800 1000 1200 1400 1600CD4
initial observationsfinal observations
Log10-transformed HIV proviral DNA as a function of CD4 and end (follow-up) plasma viremia (plasma viral load < 2.5 genomic copies/ml or > 2.5). Linear predictions are reported, along with 95% confidence intervals. Legend: rnaend = 0 means partial plasma HIV RNA suppression, rnaend = 1 means full plasma HIV RNA g p p pp psuppression; time = 0 indicates initial observation time (T0); time = 1 indicates one year of treatment (which is also close to the mean time of observation). Initial observations (timepoint T0) and final observations (timepoint T1) are reported as well.
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Cox proportional hazards regression
Figure 2
.8
1p p g
4
.6
Sur
viva
l
.2
.4
0 1 2 3 4 5
baseline log10(DNA) 10th pctilebaseline log10(DNA) 50th pctilebaseline log10(DNA) 90th pctile
years
The proportion of patients who were exempt from the relevant event (final log10 cellular HIV DNA reducedto ≤5 copies) was plotted as a function of time (years). The effect of the baseline log10 proviral HIV DNAwas indicated as the 10th, 50th, and 90th percentiles, corresponding to 269, 1838, and 6284 copies,respectively.p y
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Table 1 1
Patients were divided based on HIV plasma viraemia levels according to the results of the 2
ultrasensitive plasma viraemia method at follow-up. Two patients (1 %) were not included 3
due to the lack of ultrasensitive data. Results are expressed as median values (range) and rate 4
5
HIV RNA
<2.5 cps/ml§
HIV RNA
2.5-20 cps/ml§
HIV RNA
21-49 cps/ml§
HIV RNA
50-1000 cps/ml§
Patients No. (%) 73 (40.5) 49 (27.2) 25 (13.9) 31 (17.2)
Primary infection
Patients No. (%)*
16 (22) 1 (2) 2 (8) 2 (6.4)
CD4 count at
baseline#
296
(0-1090)
240
(10-390)
181
(6-770)
230
(0-680)
CD4 count at
follow-up#
571
(150-1418)
490
(270-941)
436
(130-1430)
520
(100-1170)
CD4 % at
Baseline
16 (0-42) 14 (0-29) 11 (2-38) 17.5 (0-33)
CD4 % at
Follow-up
30.7 (6-51) 28 (13-44) 22 (5-46) 30 (6-51)
DNA°
at Baseline
3.09
(1.34-4.84)
3.28
(1.78-3.94)
3.32
(1.96-3.80)
3.51
(2.69-4.82)
DNA°
at follow-up
1.41
(<0.69-2.89)
2.40
(<0.69-3.52)
1.77
(<0.69-3.15)
2.38
(<0.695-1.91)
DNA <5 ° (%)^ 27 (37) 7 (14.3) 3 (12) 2 (6.4)
Current Protease
Inhibitors (%)^^
24 (33) 21 (42.8) 15 (60) 17 (54.8)
Years of
follow-up
2 (0.5-5) 1 (0.5-4) 2 (0.5-4) 1 (0.5-5)
* percentage of patients with primary infection out of subiects grouped by residual viraemia. 6
# cells/mm3 7
°Cellular HIV DNA log10 copies/106 PBMCs 8
§ Plasma viraemia HIV RNA copies/ml 9
^ percentage of patients reaching DNA < 5 copies /106 PBMCs out of subiects grouped by 10
residual viraemia. 11
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^^ percentage of patients taking Protease Inhibitors out of subiects grouped by residual 12
viraemia. 13
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