Transfer occurred after a variable in vitro culture period ranging from 4–7 days after ovulation induction (OI). Following ET, serum E2/P ratios were calculated for clinical pregnancies, preclinical abortions and non-coneption cycles.239 women consecutively treated by IVF or ICSI were retrospectively analyzed and early luteal serum E2/P ratio could differentiate between clinical pregnancies and non-pregnant cycles .Receiver-operator curve analysis demonstrated that the E2/P measurements and may help improve IVF treatment outcome. Further prospective studies should be undertaken to confirm these obersveration.These retrospective data may hold prognostic value regarding endometrial receptivity as reflected by E During IVF-ET, controlled ovarian hyperstimulation results in excessive follicular development and supraphysiologic serum concentrations of E2 and P. Such derangements raised concerns about the impact of such abnormalities on the luteal phase and a possible adverse impact on endometrial tissue [2 initiates hypertrophy and hyperplasia of endometrial epithelia, but its role in the luteal phase remains poorly understood. How E2 influences endometrial synchronization and blastocyst implantation is also not well described [et al [2-prepared endometrium into a secretory tissue and creates a hospitable environment for embryo attachment [Progesterone (P) and estradiol  patient age in this study population was 32.7 ± 3.97 years (range 18–41). Fourty-nine clinical pregnancies were achieved in 239 cycles  after ET and a clinical pregnancy rate of 21.0 % per ET was determined. When stratified by diagnostic category, 106 patients had tubal disease (44.4%), 38 had endometriosis (5.4%), 13 had polycystic ovary syndrome (15.9%), 18 had unexplained infertility (7.5%), and 64 had male factor infertility (26.8%). For some patients, more than one infertility factor was assigned. ICSI was indicated for prior failed IVF fertilization(s) and male factor infertility as defined by severe semen abnormalities where semen analysis showed a sperm count of <20 M/mL. Patients were selected on the basis of a stimulation protocol from a computer-generated random number table.®, Hoechst, Frankfurt, Germany) at a dose of 0.15 mg, 3 times daily from the midluteal phase of the cylce preceding the treatment cycle followed by rFSH . Additionally, 178 women were treated with rFSH  starting on day 2 of the menstrual cycle. From day 6–7 of the index cycle, 0.25 mg of ganirelix  was administered daily as a subcutaneous injection up to and including the last day of rFSH administration. Serum concentration of E2 (pg/mL) and transvaginal ultrasound were used to monitor follicular growth. Ovulation was triggered by i.m. administration of 10,000 IU of hCG  when the mean follicular cohort diameter reached 19 mm. Ovulation induction (OI) was the beginning of the luteal phase and was designated as OI day 0. Oocyte retrieval was carried out transvaginally under ultrasound guidance 34–36 h after OI. Previous studies have described ICSI and IVF procedures in detail [pn) formation; only metaphase II oocytes were counted in IVF/ICSI cycles. Transfer was carried out 4 days after OI (OI +4 days), 5 days after OI (OI +5 days) or 7 days after OI (OI +7 days). Normally cleaved embryos were replaced under ultrasound guidance using a K-soft 5001 catheter . All patients had luteal support with Utrogestan vaginal capsules 2 × 100-mg capsules, twice a day  beginning on the day of embryo transfer. Patients with less than an E2 level <1500 pg/mL on the day of oocyte retrieval recieved only one additional luteal support of 1500 IU hCG . Venous blood samples were collected on the morning of oocyte retrieval and on the day of ET. Serum E2 and P concentrations were measured via electrochemiluminescence immunoassay "ECLIA" . For E2, inter- and intra-assay coefficients of variation on high concentration control (high E2: 1018 pg/mL) were 2.8 and 1.9%, respectively. For P (high P: 30.2 ng/mL), the inter- and intra-assay coefficients of variation were 5.5 and 2.7%, respectively. The ratio of E2/P was calculated for conception and non-conception cycles as defined below.In this study, two controlled ovarian hyperstimulation protocols were used: 61 women were treated with a conventional long protocol using a combination of intranasal buserelin  was used to test significant difference between groups. Hormonal data were log-transformed to correct for skewness prior to statistical analysis and values in the three groups were compared using the nonparametric Kruskal-Wallis test. Significance was interpreted as p < 0.05. All data were presented as mean ± SD.Statistic Package for Social Sciences  software was used for data analysis. Statistical significance was assessed using the Student From this, receiver operating characteristic (ROC) curves were developed to depict probability of true-positive results (sensitivity) as a function of false-positive results (1 -specificity). Sensitivity and specificity were calculated for all determined ratios of the decision axis and combined with the area under the curve (AUC). The AUC (sensitivity/1 - specificity) format approach was used to confirm test adequacy (AUC near 1) or inadequacy (AUC near 0.5).2 was 1174.7 ± 828.0 pg/mL (range 164–7196), and mean number of retrieved oocytes was 8.87 ± 6.09 (range 1–28). Only one case of severe OHSS was encountered. The mean number of pre-embryos replaced was 2.5 ± 0.8 (range 1–4). The clinical pregnancy rate per ET was 18.0% (OI +4 days), 21.5% (OI +5 days) and 43.3% (OI +7 days). Fourty-nine (21.0%) had a viable intra-uterine pregnancy at 8 weeks gestations, 27 (11.2%) had an abnormal pregnancy  and 163 (67.8%) failed to conceive. There was no influence of the method of fertilization (IVF or ICSI) on the outcome .The means (±SD) of various clinical parameters for clinical pregnancies, preclinical abortions and for non-conception cycles are presented in Table p = 0.005), the number of oocytes retrieved (p = 0.002), and number of fertilized oocytes (p < 0.0001). There were no significant differences in basal FSH, number of gonadotropin units (rFSH) consumed and peak E2 and P on the day of oocyte retrieval among the three groups. The use of a GnRHa long protocol or a GnRH antagonist protocol did not alter the hormonal profile dynamics, the E2/P ratio or clinical pregnancy rate.There were significant differences when related to the age of the patient , OI +5 days (p = 0.005) and OI +7 days (p = 0.0001) compared with those who had either a preclinical abortion or failed to conceive  hormonal parameters at different days of ET  and derived E2/P ratio as measured on OI +4 days, OI +5 days and OI +7 days with respect to clinical pregnancy, the AUCROC was determined with ROC analysis , on OI +5 days  and on OI +7 days  and the clinical pregnancy rate.To analyze the prognostic power of Es Figure . The are2 priming phase is required followed by P. In the pre-GnRH agonist era, the alteration of the E2/P ratio was considered a main cause of luteal-phase inadequacy and IVF failure, possibly mediated by the luteolytic action of E2 [2and P receptors are found in glandular and stromal compartements [2 on the endometrial glands by down-regulating estrogen receptors and is followed by a subsequent disappearance of P receptors [For normal endometrial morphology to occur, an Eon of E2 . The actrtements . P antageceptors .2 and P may also warrant consideration [2/P ratio at the time of embryo transfer was compared with the pregnancy outcome. Our working hypothesis of this investigation is that a high P level in combination with a low E2 level in the early luteal phase could presage failed implantation. These data suggest that the E2/P ratio on OI +4 days, OI +5 days and OI +7 days are significantly associated with clinical pregnancy rate. Interestingly, a significant higher clinical pregnancy rate could be achieved if blastocysts on OI +7 days were transferred. It seems that, blastocyst transfer allowed the identification of embryos with very high implantation potential [Many stimulation cycles in assisted reproduction are associated with failed pregnancy despite the transfer of apparently healthy and morphologically normal embryos. This suggests impairment of endometrial differentiation or receptivity in response to Ederation . In our otential , and prootential .2 and P on day of oocyte retrieval or in the early luteal  E2 and P concentrations between pregnant and non-pregnant women. The use of a single luteal E2 and P measurement to predict endometrial receptivity was not useful, although at our center the early detection of a low E2 level did help identify those who were then given supplementary hCG support for corpus luteum rescue. Additionally, a single P value in the early luteal phase was not informative for diagnosing luteal phase defect. Assessment of the E2/P ratio in the early luteal phase provided better prognostic information with relatively higher values of this ratio being associated with a healthy corpus luteum activity and successful implantation.Specifically, we identified no differences in peak E2/P ratio was not affected by the treatment protocol used (GnRH agonist vs. antagonist), findings that agree with previous work [In this study, patients received both GnRH-agonist and GnRH-antagonist stimulated cycles for controlled ovarian hyperstimulation to prevent premature LH surge. Uniform luteal support consisting of vaginal micronized progesterone starting on the day of ET was given to all patients in order to compensate for possible iatrogenic luteal phase defect . GnRH- aous work .2/P ratios and better pregnancy outcomes, whereas a high increase in P values in combination with a decrease in E2values (reflected by a low E2/P ratio) append to indicate poor reproductive outcome. Consequently, this retrospective study implies that in the latter setting the embryo will encounter a poorly receptive endometrium on the day of transfer, resulting in impaired implantation.We conclude that moderately increased P values in the early luteal phase was associated with higher E2/P ratio in relation to IVF outcome. Further study is needed to examine whether the E2/P ratios could be used as a prognostic test to predict which women will have a clinical pregnancy in the setting of advanced reproductive technologies following a COH.To our knowledge, this is the first study specifically evaluating EThe author(s) declare that they have no competing interests.


By persisting in infected cells for a long period of time, proviral HIV-1 DNA can represent an alternative viral marker to RNA viral load during the follow-up of HIV-1 infected individuals. In the present study sequential blood samples of 10 patients under antiretroviral treatment from 1997 with two NRTIs, who refused to continue any antiviral regimen, were analyzed for 16 – 24 weeks to study the possible relationship between DNA and RNA viral load.The amount of proviral DNA was quantified by SYBR green real-time PCR in peripheral blood mononuclear cells from a selected group of ten patients with different levels of plasmatic viremia .Variable levels of proviral DNA were found without any significant correlation between proviral load and plasma HIV-1 RNA levels. Results obtained showed an increase or a rebound in viral DNA in most patients, suggesting that the absence of therapy reflects an increase and/or a persistence of cells containing viral DNA.Even though plasma HIV RNA levels remain the basic parameter to monitor the intensity of viral replication, the results obtained seem to indicate that DNA levels could represent an adjunct prognostic marker in monitoring HIV-1 infected subjects. Many papers have clearly demonstrated that HIV-1 RNA plasma viral load quantitative determination is a pivotal parameter to monitor viral replication and the effectiveness of HAART therapy . In addiRecent studies also indicate that viral replication persists even in individuals with prolonged suppression of plasma HIV-1 RNA levels to fewer than 50 copies/ml ,14-16, cSince a growing number of studies involving quantification of cellular HIV-1 DNA acknowledge the importance of accurate quantification of proviral DNA in peripheral blood cells for monitoring diseases progression, we selected a small but peculiar group of patients, who decided to interrupt antiretroviral therapy, irrespective of current guidelines  and despTen HIV-1 infected adults under antiretroviral treatment since 1997 with two NRTIs  were selected for this study. All these patients refused to continue any antiviral regimen despite an assessed virologic failure  and were followed-up monthly for a variable period ranging from 16 to 24 weeks up to the moment in which they agreed to begin a new therapeutic protocol. Sequential blood samples were obtained at baseline (time 0: voluntary therapy interruption) and every four weeks (time 1-time 7) and analyzed for viral load (RNA and DNA), and CD4 levels. The baseline characteristics of the patients included in the study are shown in Table All the whole blood samples were centrifuged at 2500 rpm for 20 min and plasma was stored at -80°C until use. Plasma was analyzed for HIV-1 RNA viral using the Quantiplex HIV-RNA-3.0 assay , according to the manufacturer's instructions. The amount of HIV RNA levels was expressed as copy number per ml of plasma and the lowest detection limit of the assay was 50 copies/ml.6 PBMC were prepared and stored at -80°C. DNA was extracted and purified from each PBMC pellet by DNAeasy tissue kit (Qiagen) following the manufacturers' instructions. The pGEMBH10 HIV plasmid  at the end of observation period (p = 0.014) (Table 10 to more than 1 log10 at the end of observation period (p = 0.00).As expected, therapy interruption determined a significant increase in RNA viral load in all HIV-1 seropositive patients enrolled in the study. In particular, all patients' plasma showed a significant (Mann-Whitney test p = 0.036) increase in viral load already one month after therapy interruption (time 1) showing a median value of 1.7 × 104 4. log10 inIn parallel experiments, we quantified proviral DNA load in PBMC isolated from patients' whole blood sequential samples at fixed times after therapy suspension. The median number of samples available for each patient was five [1 month after the therapy suspension (time 1) and then each month up to the end of observation period], ranging from two to seven. The median follow-up was 5.5 (4–7) months.10) higher value only in samples from patients N°1 and N°10 (from 3.2 log10 to 3.7 log10 and from 2.7 log10 to 3.4 log10 respectively), sequential PBMC samples obtained from patient N°9 exhibited a clear tendency to increase (and from 3.1 log10 to 3.5 log10 copy of HIV-1 DNA per 106 PBMCs respectively) redoubling the DNA content. Moreover, most of the other samples obtained from patients N°2, N°3, N°4, N°5 and N°6 showed a swinging course. After an apparent decline in proviral DNA content during the follow-up, in the latest samples a moderate increase in proviral DNA load was observed in PBMC from all patients. In contrast, a decrease of HIV-1 proviral DNA content was noticed from a baseline value of 1.3 × 103 copies per ml (3.1 log10) to 5.5 × 102 HIV-1 DNA copies per ml (2.7 log10) and from 9.2 × 102 (2.9 log10) to 3.0 × 102 (2.4 log10) in patients N°4 and 7 only.The majority of patients showed a fluctuating trend in DNA viral load. Three patients  showed an increase in DNA viral load detectable from the first through to the last available sample. Even though DNA amount reached a significantly  confirming our previous data .All the patients enrolled in the study showed a CD4 reduction during the follow-up. All patients, except two (Patients N°1 and N°2), interrupted therapy with a level of CD4 cells >400 cells/mmc and, as expected, showed a sharp  decrease or a moderate decline  at the end of our observation period. No correlation was found  between the course of DNA viral load and CD4 levels, but high RNA levels were significantly associated with lower CD4 counts, demonstrating a significant inverse correlation between CD4+ cell counts and HIV-1 RNA levels (p = 0.001).During recent years, planned therapy interruption has been entertained in specific clinical situations even though the potential role of this choice with respect to the balance between risk of disease progression and potential benefits remains to be elucidated. Our study focused on a peculiar group of patients who voluntary opted to suspend antiretroviral therapy for a variable period of time, ranging from five to seven months, despite of virologic failure. Our follow-up ceased when patients agreed to a new therapeutic protocol.et al. [Our study aimed to evaluate the virologic evolution of these subjects focusing on DNA proviral load course, since accurate quantification of HIV-1 DNA in peripheral blood cells is an important parameter for monitoring disease progression and predicting the clinical outcome of infection ,27-29. Set al.  recentlyCurrent data on course of DNA viral load during infection are inconclusive -9, but mOur results, obtained from patients, therapy-free during the virolgical follow-up, showed a viral rebound, one month after therapy suspension, assessed by plasma RNA values in all patients. The analysis of HIV-1 DNA proviral content displayed a clear increase from the baseline value in three patients, confirming that an active viral replication results in elevated viremia (HIV-1 RNA load) and in an increased number of cells containing viral DNA ,28. AlsoFinally, in contrast with other patients, two subjects showed a clear HIV-1 DNA proviral decrease over time, in the absence of therapy and a steady HIV-1 RNA viral load detectable in plasma samples. In both cases a HIV-1 DNA proviral decline due to a long lasting effect of therapy could be ruled out, since both patients showed high levels of viral replication by increasing value of HIV-1 RNA viral load over time. In an attempt to explain the course of HIV-1 DNA proviral in these subjects, we had to take into consideration that our assay, a SYBR green based real time PCR measures both integrated and unintegrated HIV-1 DNA form on PBMCs. There is evidence that only a fraction of integrated and unintegrated HIV-1 DNA is replication competent . Hence, Despite contrasting reports on the meaning of DNA proviral content in HIV-1 seropositive patients ,5,9-12, Only careful evaluation of virological and immunological markers is necessary to fully characterize the course of HIV-1 infection and to provide a more complete laboratory-based assessment of disease progression. However, the availability of a new standardized assay such as DNA proviral load will be important to assess the true extent of virological suppression in patients with non-quantifiable plasma viral loads and to verify the efficacy of new immune-based therapies aimed at purging HIV-1 DNA reservoirs. Although the biological meaning of DNA proviral load in PBMCs is not yet clear, several studies ,3,6,10 sThe author(s) declare that they have no competing interests.MCR and DG conceived and designed the study. FS and PV developed the HIV-1 DNA real time and performed all the experimental work. LS and FG provided blood samples and clinical information on the patients enrolled in this study. MCR drafted the manuscript and DG reviewed it. All authors contributed to the final version of manuscript, read and approved it.The pre-publication history for this paper can be accessed here:


Current antiretroviral therapy is effective in suppressing but not eliminating HIV-1 infection. Understanding the source of viral persistence is essential for developing strategies to eradicate HIV-1 infection. We therefore investigated the level of plasma HIV-1 RNA in patients with viremia suppressed to less than 50–75 copies/ml on standard protease inhibitor- or non-nucleoside reverse transcriptase inhibitor-containing antiretroviral therapy using a new, real-time PCR-based assay for HIV-1 RNA with a limit of detection of one copy of HIV-1 RNA. Single copy assay results revealed that >80% of patients on initial antiretroviral therapy for 60 wk had persistent viremia of one copy/ml or more with an overall median of 3.1 copies/ml. The level of viremia correlated with pretherapy plasma HIV-1 RNA but not with the specific treatment regimen. Longitudinal studies revealed no significant decline in the level of viremia between 60 and 110 wk of suppressive antiretroviral therapy. These data suggest that the persistent viremia on current antiretroviral therapy is derived, at least in part, from long-lived cells that are infected prior to initiation of therapy. Combination antiretroviral therapy is effective in reducing, but not eliminating, HIV-1 replication. Residual viremia during suppressive antiretroviral therapy may arise from a number of sources, including reservoirs of long-lived virus-producing cells, or ongoing complete cycles of viral replication. Here, we used a new, more sensitive assay of HIV-1 RNA to measure residual viremia in a large cohort of patients with prolonged suppression on antiretroviral therapy. We found a persistent, stable level of viremia in patients on prolonged therapy that correlated with pretherapy levels of HIV-1. Over 80% of patients had viremia ≥1 copy/ml plasma, and the level of viremia was independent of the drug regimen patients were taking. These data strongly suggest that persistent viremia on antiretroviral therapy is likely derived from reservoirs of long-lived virus-producing cells that are not affected by currently available drugs that target new cycles of viral replication. New antiviral strategies that eradicate this reservoir will be necessary to cure HIV-1 infection. Infection with HIV-1 results in progressive immunodeficiency and death from opportunistic infection or cancer. Current antiretroviral therapy is effective in suppressing plasma viremia to levels below the detection limit of FDA-approved assays (50–75 copies HIV-1 RNA/ml), restoring immune function, and reducing morbidity and mortality. Antiretroviral therapy does not cure HIV-1 infection, however, and, at a minimum, eradication of HIV-1 infection will require complete suppression of its replication as well as elimination of latent viral reservoirs. Although some reports have shown the persistence of low-level viremia in patients on suppressive therapy , the detWe first measured plasma HIV-1 RNA with both an FDA-approved assay  and SCA in three patients initiating antiretroviral therapy. As expected –13, therp = 0.56) or overall distribution of values (p = 0.82). Plasma HIV-1 RNA was below the limit of quantification by SCA in about 17% of patients .These initial observations suggested that low-level viremia can persist for years in patients receiving suppressive antiretroviral therapy. To investigate this phenomenon in a larger patient population and to compare the effects of different treatment regimens, we analyzed specimens from study M98–863, a Phase III randomized clinical trial comparing lopinavir/ritonavir (LPV/r) and nelfinavir (NFV), each in combination with stavudine and lamivudine, in previously antiretroviral-naïve HIV-1-infected individuals . We studp = 0.22); specifically, the mean values were not significantly different (p = 0.17) and there was no difference between efavirenz- and nevirapine-containing regimens (p = 0.29).We also determined the distribution of HIV-1 RNA levels in 28 patients enrolled in National Institutes of Health (NIH) studies whose viremia was suppressed to <75 copies/ml on non-nucleoside reverse transriptase inhibitor (NNRTI)-containing regimens. As shown in p < 0.001) and a median decrease between pretherapy and week 60 HIV-1 RNA values of 14,250-fold . The correlation coefficient (r2 = 0.17) suggests pretherapy viral RNA level accounts for 17% of the variability in stable viremia on therapy, and that other factors, such as time since infection, may contribute to the level of on-therapy viremia. No significant differences between treatment arms were detected in median HIV-1 RNA reduction, and additional analyses (unpublished data) indicated that both the magnitude of HIV-1 RNA reduction and the level of persistent viremia were not associated with pretherapy CD4+ T-cell count, change in CD4+ T-cell count on therapy, age at enrollment, or gender.We investigated virologic, immunologic, demographic, and clinical parameters for correlates of persistent viremia in samples from both arms of the M98–863 trial. Comparison of the HIV-1 RNA level by SCA at week 60 to pretherapy RNA levels  showed aTo examine the stability of the low level of viremia in the M98–863 study, we next performed a longitudinal analysis of HIV-1 RNA after week 60. Of the 145 patients with pretherapy samples who had suppression of plasma HIV-1 RNA between weeks 24 and 60, 117 had samples available and continuous suppression  was more than 10-fold below the limit of detection of FDA-approved HIV-1 RNA assays. Viremia was suppressed to less than 0.6 copies/ml in approximately one-fifth of patients, nearly 100-fold lower than the detection limit of FDA-approved assays. As such, SCA permitted a more accurate estimate of the maximal effect of antiretroviral therapy. Comparison of pre- and post-therapy levels revealed a median reduction in HIV-1 RNA of more than 14,000-fold. After this maximal reduction in viremia occurred, however, HIV-1 RNA levels remained stable with no significant decline between the first and second years of therapy.In previous studies, investigators using smaller datasets have reported slow declines in plasma HIV-1 RNA corresponding to half-lives of infected cells of 6 mo ,15. Our http://clinicalstudies.info.nih.gov/cgi/protinstitute.cgi?NIAID.0.html). Information from such trials is essential in designing future antiretroviral strategies to fully suppress viremia.Three sources of persistent viremia have been proposed: (i) ongoing cycles of viral replication in the presence of antiretroviral drugs because of inadequate drug inhibitory potency; (ii) production of HIV-1 from sanctuary sites into which antiretrovirals do not penetrate sufficiently; or (iii) reservoirs of long-lived cells infected prior to the initiation of therapy with production of HIV-1 from integrated proviruses, but without complete cycles of replication occurring because of blockade by antiretroviral drugs ,15–17. T50 for wild-type HIV by a considerably greater margin than observed with NFV. Consequently, individual pharmacokinetic variability and adherence lapses may be more likely to result in loss of suppression with NFV-based regimens compared to LPV/r-based therapy. Similar levels of persistent viremia in patients undergoing NNRTI-containing therapy for prolonged periods (mean = 111.2 wk) were noted even though the genetic barrier to NNRTI is likely to be low; again, early drug failures due to pre-existing mutations were likely excluded from selection. The presence of persistent viremia has a number of important implications for development and application of antiretroviral therapies. Few patients on antiretroviral therapy have “undetectable” viremia if sufficiently sensitive assays are employed, and greater suppression of viremia can probably not be achieved using current inhibitors of HIV-1 replication because persistent viremia likely arises, at least in part, from chronically infected cells. New therapeutic strategies will be needed to eliminate persistent viremia and its source.This study analyzed only those participants in the M98–863 study whose viremia remained suppressed to <50 copies/ml for 60–110 wk. As demonstrated in prior intent-to-treat analyses of M98–863, LPV/r had superior efficacy compared to NFV as judged by Kaplan-Meier analysis of plasma HIV-1 RNA rebound to greater than 400 copies/ml through 96 wk ,25–27. TPlasma samples were obtained from stored specimens from patients with plasma HIV-1 RNA <50 copies/ml in the M98–863 trial and from patients on treatment at the NIH Clinical Center as follows.n = 326) with NFV  (n = 327), each in combination with stavudine and lamivudine, in previously antiretroviral-naïve HIV-1-infected patients [n = 237). From this group, a subset of 163 participants treated at investigational sites in North America was identified for SCA testing. Of these, six were excluded for lack of archived samples and 12 for inefficient amplification of baseline sample. Pretherapy samples of 145 participants were amplifiable by SCA  . All patients were taking Department of Health and Human Services (DHHS) guideline-approved antiretroviral regimens containing efavirenz (n = 22) or nevirapine (n = 6) and were either sampled frequently by bDNA or SCA following initiation of therapy or had been on therapy for 20 to >250 wk (mean 111.2 wk) prior to SCA sampling. All patients were well and without history of recent intercurrent illness at the time of phlebotomy, had hemoglobin levels ≥12 g/dl, and provided informed consent for phlebotomy and for research sample storage.Patients taking NNRTI-containing regimens with suppressed plasma HIV-1 RNA levels by FDA-approved testing were also recruited from treatment studies performed at the NIH Clinical Center . For patients taking NNRTI-containing regimens, plasma HIV-1 RNA was quantified using the bDNA-based VERSANT HIV-1 RNA assay version 3.0 according to manufacturer's specifications  in a manufacturer-certified site using the semiautomated Bayer System 340 unit. Versant HIV-1 RNA version 3.0 is approved by the FDA for clinical use with a limit of quantification of 75 copies HIV-1 RNA/ml plasma, although our reported experience with this assay indicates a quantification limit of 50 copies/ml [Study M98–863 used the PCR-based Amplicor HIV-1 MONITOR assay version 1.0 for plasma HIV-1 RNA quantitation, performed according to manufacturer's specifications  were considered to be equal to the assay limit. Sensitivity analyses using other imputation methods did not alter results meaningfully.Figure S1(21 KB PDF)Click here for additional data file.Figure S2n = 145, diamonds) were highly correlated ; corresponding participants were included in subsequent analyses.Pretherapy samples from 157 participants were analyzed by both assays. 12 samples (triangles) had SCA values significantly lower than Amplicor and were excluded from further analysis. Remaining samples ((32 KB PDF)Click here for additional data file.


No therapies have been proven to persistently improve the outcome of HTLV-I-associated myelopathy. Clinical benefit has been reported with zidovudine and with lamivudine in observational studies. We therefore conducted a randomised, double blind, placebo controlled study of six months combination therapy with these nucleoside analogues in sixteen patients.Primary outcomes were change in HTLV-I proviral load in PBMCs and clinical measures. Secondary endpoints were changes in T-cell subsets and markers of activation and proliferation.Six patients discontinued zidovudine. No significant changes in pain, bladder function, disability score, gait, proviral load or markers of T-cell activation or proliferation were seen between the two arms. Active therapy was associated with an unexplained decrease in CD8 and non-T lymphocyte counts.in vivo, inadequate intracellular concentrations of the active moiety or the contribution of new cell infection to maintaining proviral load at this stage of infection may be relatively small masking the effects of RT inhibition.Failure to detect clinical improvement may have been due irreversible nerve damage in these patients with a long clinical history and future studies should target patients presenting earlier. The lack of virological effect but may reflect a lack of activity of these nucleoside analogues against HTLV-I RT  The first identified human retrovirus, Human T-cell Lymphotropic Virus Type I (HTLV-I) , was iniThe onset of HAM is usually sub-acute and the initial presenting symptoms may relate to the urinary bladder, bowels or sexual function as well as gait disturbance or back pain. Diagnosis is often delayed until the more characteristic constellation of combined symptoms appears some months or years later. The most rapid progression is usually seen within the first two years with the neurological deficit varying from mild gait impairment to paraplegia. Thereafter some patients remain relatively stable whilst slow deterioration compounded by physiological aging is observed in others. In our experience approximately half of all patients become wheel-chair dependant and although many patients with HAM have a normal life span, premature deaths directly related to HAM occur.The pathogenesis of HAM is not fully understood but several observations point to potential therapeutic approaches. An association between proviral load and disease has been repeatedly found with proviral load, as measured by HTLV-I DNA copies in peripheral blood mononuclear cells (PBMCs), about ten fold higher in patients with HAM than in asymptomatic carriers,8 and thThe literature on the specific management of HAM consists mostly of small, uncontrolled studies and cohort data. Conflicting results have been obtained in studies of corticosteroids therapy. InterfeWe report here the results of a randomised, double blind, placebo controlled study of the combination of zidovudine and lamivudine to determine their safety and efficacy with medium term (six to twelve months) usage.Sixteen patients were recruited to the study, twelve in London and four in Kagoshima. Eight were randomly assigned to each study arm. Baseline demographics, clinical and laboratory characteristics of the participants are shown in Table rd month to 25.5% at the end of the 6th month. There was little detectable difference in returned tablet counts between the two arms or between the first, randomised and second, open phases of the study.Mean red cell volume rose during the first 24 weeks of the study in the active arm. At week 24, 100% of patients in the active arm and 0% in the placebo arm had a change in MCV from baseline greater than 3 within-person SDs. During the second 24 weeks of the study a similar rise in MCV was seen in the placebo arm at week 48, 75% of patients in each arm had a change in MCV from baseline greater than 3 within-person SDs. There was good consistency between lamivudine and zidovudine returned tablet counts but the average number of tablets returned in the previous month rose from 9.5% at the end of the 3During the first 24 weeks of the study no participants in the placebo arm discontinued therapy. In the active arm one participant, whose disease had progressed from first symptom to bed-bound in the 9 months prior to study entry, was un-blinded at week 8 because of continuing deterioration. Interferon-α was added to the active compounds until week 20 when zidovudine was discontinued following the development of autoimmune haemolytic anaemia. During the second 24-week period of open therapy 5 participants previously on placebo discontinued zidovudine, one within 4 weeks, with gastrointestinal symptoms, the remainder after 16 – 20 weeks of therapy. Two had anaemia, one necessitating blood transfusion, one complained of drowsiness and paraesthesia and one of lethargy. One participant from the active therapy arm elected to discontinue therapy at week 40 when antibiotics were prescribed out of study. No side affects attributable to lamivudine were reported. There were no significant biochemical abnormalities.No significant changes in pain score, urinary frequency or nocturia were seen between the active arm and the placebo arm during the study Table  nor with10/100,000 PBMCs). No significant change in proviral load was seen during the first 24 weeks of the study in the active arm compared with the placebo arm (mean difference (SE): 0.02 (0.17), p = 0.92) (Table Laboratory markers of efficacy: The median (IQR) HTLV-I proviral load at baseline was 4.2 (2.6–7.5) copies per 100 PBMCs (mean (SD) 3.7 (0.4) log6/L in the placebo arm but decreased by 159 × 106/L in the active arm, a mean difference of 307 × 106/L  (Table 6/L followed by a decrease of 71 × 106/L (mean difference (SE): 219 (73), p = 0.02) . A similar trend was seen by comparing active with placebo therapy in the placebo arm, although not statistically significant.At baseline, the total lymphocyte, CD3, CD4 and CD8 cell counts were normal. During the first 24 weeks the mean total lymphocyte counts (AUCMB) increased by 148 × 10At baseline CD69 was expressed on average on 8%, CD71 on 8% and CD25 on 9% of lymphocytes. Compared with placebo no significant changes were seen in these markers of activation (CD69) and proliferation (CD71) nor in the expression of the IL-2 receptor (CD25) Tables  and 4 duSince monotherapy with nucleoside analogues had been reported to reduce HTLV-I viral load, the inhibition of HTLV-I reverse transcriptase by two nucleoside analogues, zidovudine and lamivudine was thought to be more likely to cause a sustained decrease in HTLV-I viral burden than either drug alone. A decrease in HTLV-I viral DNA and thus a presumed decrease in viral antigen burden has been associated with a reduction in HTLV-I specific CTL lymphocyte activity.Since this activity is postulated to contribute to HAM pathogenesis anti-HTLV therapy might in turn delay progression or cause clinical improvement.HAM is a rare disease in the UK with only 10 – 12 cases diagnosed annually. A similAlthough the basic study design was double-blind and placebo-controlled a second open-therapy phase was incorporated, with all study participants offered open therapy for a further six months, to maximise the likelihood of detecting significant clinical and laboratory changes. The study was not un-blinded until the last participant had completed twelve months of therapy. In this way the safety of zidovudine plus lamivudine could be studied for a total of twelve months exposure whilst those originally randomised to the placebo arm acted as their own control for the second six months of active compound in a non-randomised comparison.The primary outcome measures of the study were the clinical and virological effects of zidovudine in combination with lamivudine. The failure to detect any clinical improvement after up to 12 months of therapy may have been due to the long history of HAM among the participants who had an average duration of symptoms of 10.1 years. After this prolonged time the neurological damage may have been irreversible. However, the stability of the HTLV-I viral DNA load, which was expected to fall during the study, leaves the possibility that agents that effectively reduced viral burden, might result in clinical improvement even in longstanding disease.The persistence of high viral load during therapy could be due to a lack of activity of these nucleoside analogues against HTLV-I RT, inadequate intracellular concentrations of the active triphosphate metabolite, or it may be that reverse transcription is not important in maintaining HTLV-I proviral load at this stage of infection. Thus, whilst a reduction in HTLV-I viral DNA during therapy with lamivudine has been documented in vivo, there are now conflicting reports of its activity against HTLV-I RT in vitro -26. ConvChanges in the lymphocyte markers of activation and proliferation might have been expected with a reduction in HTLV-I proviral load. In the absence of such an effect the observed, statistically significant, changes in total, CD4, CD8 and non-CD3 lymphocytes are of interest albeit unexplained.The data from this study do not provide evidence of improvement in the clinical state of patients with HAM in the medium term following treatment with the combination of zidovudine and lamivudine but the treatment was well tolerated with no unexpected side effects. New, controlled studies of both anti-viral and anti-inflammatory agents are urgently required for patients with this chronic, disabling disease. International co-operation will be necessary to accelerate progress.10 copies of HTLV-I DNA per 100 PBMCs, was powered to detect a difference in average change from baseline up to 24 weeks (as measured by area under the curve minus baseline – AUCMB) of 1 log10 copy with 90% power and 5% probability of type 1 error. Therapeutic safety was determined by monitoring standard haematological and biochemical parameters according to National Institute of Health, Division of AIDS criteria[Patients were screened two weeks prior to baseline and eligibility criteria checked. Recruitment was by the study physicians at each site. The eligibility form was submitted with a request for a trial number to the coordinating centre at the Medical Research Council. Trial numbers had been randomly allocated, stratified by site, to active or placebo and this code was held, sealed and secure, at the MRC. Trial therapy was stored in site pharmacies and labelled by trial name and trial number only. The trial coordination staff at the MRC issuing the trial number, the pharmacy, the clinician and the patient were all blinded to the allocation of study arm. Trial numbers were transmitted by facsimile to the study centres. Recruitment commenced 8 November 1999. Eligible patients were randomised to start 24 weeks treatment with zidovudine 300 mg plus lamivudine 150 mg twice daily (the active arm) or matching zidovudine and lamivudine placebo tablets (the placebo arm). This was followed by 24 weeks of open therapy with the active compounds for all study participants. Last follow up was completed 30 July 2002. The primary endpoints were virological – change in HTLV-I viral DNA from baseline to 24 weeks and clinical – changes in gait, disability, pain and bladder function. The secondary endpoints were immunological – changes in CD4, CD8, CD25, CD69 and CD71. The study, based on recruiting twelve patients in each arm and assuming a standard deviation of 0.78 log  criteria.rd Department of Internal Medicine, University of Kagoshima, Kagoshima, Japan. At each site the local research ethics committee approved the study and written informed consent was obtained in the local language.Patients were eligible for the study if they had clinical evidence of HAM according to WHO criteria , had conParticipants underwent a full neurological examination at weeks 0, 24 and 48 and if new symptoms were reported. Participants were reviewed monthly with additional visits at weeks 1, 2, 25 and 26. At each visit a fixed distance walk was timed and the degree of walking aid documented for patients able to walk. Disability was graded 1 – 13 as described by Osame . Pain waFull blood counts, biochemical profile, quantitative HTLV-I viral DNA and lymphocyte phenotypic assays were performed at each study visit. The clinical investigators were blinded to the mean red cell volumes although these were available for the end of study analysis. Full blood counts were measured on a Coulter LH750 Analyzer . The renal, liver and bone chemistry assays were performed on an AU2700 Olympus Analyser . HTLV-I viral DNA was quantified by real-time PCR as previously described. . In addi10 transformed prior to analysis. Data were analysed as average change from baseline to a given time point as measured by AUCMB using the T-test. All analyses were performed as randomised regardless of changes to study treatment during follow up. The study was funded by the departments of the participants. GlaxoSmithKline generously provided zidovudine and lamivudine for the study and matching placebos.Data from clinical record files were entered into Oracle databases and checked manually and by computer consistency checks. Analysis text files were created from the database and imported into STATA for statistical analysis. Baseline values of laboratory tests were calculated as the mean of screening and week 0 results. Week 0 data were taken as baseline for all clinical measurements. Time was measured from the start of trial treatment and for all measurements during follow-up data from the closest date to the target assessment week, within a window of ± 2 weeks, were used. HTLV-I proviral load values were converted to copies per 100,000 PBMCs and logAUCMB Area under the curve minus baselineCD Cluster of DifferentiationCTL Cytotoxic T- LymphocyteDNA Deoxyribonucleic acidHAM HTLV-I-associated myelopathyHTLV-I Human T-cell Lymphotropic Virus type IIFN-γ Interferon-gammaIL-2 Interleukin – 2IQR Inter-Quartile RangeMCV Mean cell volumeMDR Multi-drug ResistancePBMC Peripheral blood mononuclear cellSD Standard DeviationSE Standard ErrorTNF-α Tumour necrosis factor – alphaWHO World Health OrganisationGraham P Taylor has received honoraria for teaching and travel grants from GlaxoSmithKline plc. No other conflicts of interest are declared.The study was conceived and designed by GPT, AB, JNW & CRMB; conducted by GPT, PG, YF, AB, AM, NN, PR, KU & MO, analysed by HG & AB and the manuscript was written by GPT, HG, AB, CRMB & JNW


Hydroxyurea (HU) is an immunomodulatory agent that has been documented to enhance the antiretroviral activity of nucleoside reverse transcriptase inhibitors, such as abacavir (ABC) and didanosine (ddI), and would be expected to improve virologic efficacy.A 48-week, phase IV, multicenter, open-label, proof-of-concept clinical trial was conducted to evaluate second-line, protease inhibitor (PI)-sparing therapy with ABC/efavirenz (EFV)/ddI plus HU or without HU in HIV-infected subjects failing to achieve HIV-1 RNA ≤ 400 copies/mL after ≥ 16 weeks of treatment with lamivudine/zidovudine or lamivudine/stavudine, plus 1 or 2 PIs. Subjects were assigned to ABC (300 mg twice daily)/ EFV (600 mg once daily)/ ddI (400 mg once daily) plus HU (500 mg twice daily) (n = 30) or this regimen without HU (n = 24).10 copies/mL and CD4+ cell count was 345 cells/mm3. A similar percentage of subjects in the non-HU arm (58%) and HU arm (53%) completed the study. Intent-to-treat: missing = failure analysis showed no differences in proportions of subjects in the non-HU and HU arms achieving undetectable plasma HIV-1 RNA levels at week 24 . Median change from baseline in CD4+ cell count in the non-HU and HU arms at week 48 was +114 cells/mm3 and -63 cells/mm3 (P = 0.007), respectively. Both regimens were generally well tolerated, although more subjects in the HU arm withdrew prematurely from the study due to adverse events (23% vs 4%). Four cases of possible ABC-related hypersensitivity were observed.Baseline mean HIV-1 RNA was 3.86 logABC/EFV/ddI was an effective and well-tolerated second-line regimen for nucleoside/PI-experienced HIV-infected subjects. The addition of HU blunted the CD4+ cell response, did not appear to enhance antiviral activity, and resulted in more treatment-limiting adverse events. Combination antiretroviral therapy containing protease inhibitors (PIs) has contributed substantially towards delaying progression of HIV infection and decreasing morbidity and mortality ,2. HowevStudies in which the NRTI abacavir (ABC) was substituted for a PI have shown maintenance of virologic suppression and CD4+ cell elevation, with reduction in plasma lipid levels . Substitin vitro between HU and didanosine (ddI)  400 copies/mL) and the Roche PCR assay Amplicor HIV-1 MONITOR UltraSensitive Version 1.0 (LLOQ 50 copies/mL) . CD4+ cell counts were determined by flow cytometry. Urinalysis was performed at baseline and at weeks 4, 12, 16, 24, and 48.Clinical adverse events and laboratory abnormalities were assessed and graded according to the standardized AIDS Clinical Trials Group (ACTG) toxicity grading scales wherever possible (grade 1 or mild to grade 4 or severe). In cases of suspected HU-related Grade 2 or higher hematologic toxicities, hyperamylasemia, or neuropathy, HU was discontinued until the toxicity returned to equal to or less than Grade 2. HU was restarted at a reduced dose and could be subsequently increased to full dose. If amylase levels did not decrease following interruption of ddI, all study medications were to be interrupted. Persistent, recurrent, or Grade 3 or higher hematologic toxicities, hyperamylasemia, or neuropathy required permanent discontinuation of HU. Doses of ddI could be replaced with an alternate NRTI. If pancreatitis was diagnosed, all study drugs were to be permanently discontinued and the subject discontinued from the study, with or without elevated amylase levels. If a subject presented with symptoms consistent with a possible ABC-related hypersensitivity reaction, including fever, skin rash, fatigue, and gastrointestinal symptoms such as nausea, vomiting, diarrhea, or abdominal pain, therapy with ABC was permanently discontinued.The primary efficacy endpoint was the proportion of subjects achieving plasma HIV-1 RNA levels <400 copies/mL at weeks 24 and 48. Secondary efficacy endpoints assessed at weeks 24 and 48 included the proportion of subjects achieving plasma HIV-1 RNA <50 copies/mL, change from baseline in HIV-1 RNA and CD4+ cell count, and proportion of subjects with at least a 50-cell increase in CD4+ cell count. During the 48-week study, assessments were made of the time to virologic failure and time to treatment failure. Virologic failure was defined by any of the following: HIV-1 RNA >400 copies/mL by week 24 of randomized treatment or repeated detection (>400 copies/mL) after initial suppression to undetectable levels (<400 copies/mL) or a 3-fold or greater increase in plasma HIV-1 RNA level from the nadir at week 8 or later not attributable to intercurrent infection or vaccination. Treatment failure was defined by one or more of the following: virologic failure, toxicity or other treatment-related withdrawal, or clinical disease progression .10 copies/mL) and change from baseline CD4+ cell count were analyzed using the two-sample t-test and Wilcoxon sum rank test, respectively. Time to virologic failure and time to treatment failure were analyzed using the Kaplan-Meier method and compared between the treatment arms using the log-rank test. The study was not powered to show statistical differences between the treatment arms. Differences were deemed statistically significant if the P-value was <0.05.Subjects were randomly allocated to the HU and non-HU arms. Within the HU arm, subjects were randomized either to treatment with HU starting at study baseline or to HU starting at 8 weeks post-baseline. Randomization was stratified by screening HIV-1 RNA . The total number of subjects originally planned for this study was 150 (80 in the HU arm [40 starting HU at baseline and 40 at 8 weeks]) and 70 in the non-HU arm. However, very slow enrollment necessitated participation by fewer subjects. The proportions of subjects achieving HIV-1 RNA <400 copies/mL and <50 copies/mL at week 24 was calculated using an intent-to-treat: missing = failure (ITT: M=F) analysis. This analysis, which included all randomized subjects, regarded as a treatment failure any subject with missing values or who did not initiate treatment, changed treatment, or prematurely discontinued randomized treatment for any reason. The ITT: M=F analysis was not performed at week 48 because of potential bias due to early termination of the study after the last subject completed 24 weeks. An ITT: observed analysis was performed at both week 24 and week 48, which included all data from subjects seen at each specific visit. Comparisons of proportions were made using the Cochran Mantel Haenzel test. Change from baseline and average area under the curve minus baseline (AAUCMB) in HIV-1 RNA , and a higher percentage classified CDC Category A in the HU arm (63% vs 38%); one-fifth of the subjects in each treatment arm were Category C. Prior to the study, the most frequently used NRTIs in each arm had been lamivudine, d4T, and the lamivudine 150 mg/zidovudine 300 mg combination tablet , and the most frequently used PIs had been nelfinavir mesylate and indinavir sulfate.Baseline demographic and disease characteristics were similar between the treatment arms Table . Most ofOf the subjects in the HU arm, 17 started HU at baseline and 13 at week 8. A similar percentage of subjects in the non-HU arm (58% [14/24]) and HU arm (53% [16/30]) completed the study. The reasons for premature withdrawal from the study are given in Table P = 0.899, Fig. P = 0.081, Fig. P = 0.512).At week 24, the proportion of subjects in the non-HU and HU treatment arms who achieved HIV-1 RNA <400 copies/mL was not significantly different, according to the ITT: M=F analysis . No significant differences between the treatment regimens were observed in proportions of subjects achieving HIV-1 RNA <400 and <50 copies/mL in the protocol-specified subgroups of subjects with baseline HIV-1 RNA >10,000 copies/mL or between 400–10,000 copies/mL (data not shown). Median decrease in HIV-1 RNA from baseline tended to be greater in the HU arm than the non-HU arm at week 24 , but not week 48 . Median AAUCMB in HIV-1 RNA in the HU and non-HU arms was not different at week 24  or week 48 .Results with the 50-copy/mL assay paralleled those with the 400-copy/mL assay. At week 24, the proportion of subjects in the non-HU and HU treatment arms who achieved HIV-1 RNA <50 copies/mL was not significantly different in the ITT: M=F analysis , although the non-HU arm demonstrated slightly longer survival time to treatment failure than the HU arm , respectively, at week 24, and +114 cells/mm3 and -63 cells/mm3 (P = 0.007), respectively, at week 48. At week 48, the median CD4+ cell count was 403 cells/mm3 in the non-HU arm and 249 cells/mm3 in the HU arm (P = 0.168). The proportion of subjects achieving at least a 50-cell increase above baseline in CD4+ cell counts was greater in the non-HU arm at week 24  and week 48 .CD4+ cell counts increased steadily and modestly over 48 weeks in the non-HU arm, whereas they decreased slightly in the HU arm as a whole, fell markedly below baseline in subjects who started HU at the beginning of the study, and increased modestly between Weeks 8 and 40 in subjects who started HU at study week 8 Fig.  Median cThe number of subjects experiencing adverse events was similar in both treatment arms. Grade 1–4 drug-related adverse events reported by ≥ 5% of subjects are presented in Table In this prospective proof-of-concept clinical trial, combination therapy with ABC/EFV/ddI maintained modest suppression of HIV-1 RNA levels and increases in CD4+ cell counts through 48 weeks of therapy in a significant proportion of subjects who previously failed to respond to their initial NRTI/PI-containing antiretroviral regimens. Thus, this study showed that changing from a PI-based regimen to an NNRTI-based regimen for virologic failure is associated with a favorable outcome in some patients. While cross-study comparisons are limited by differences in subject populations, comparisons of the virologic findings of NZTA4008 with those from studies in subjects with similar baseline HIV disease characteristics and antiretroviral experience can suggest the relative therapeutic usefulness of ABC/EFV/ddI in this subject population. Thus, ABC/EFV/ddI resulted in more subjects achieving undetectable HIV-1 RNA levels and a more pronounced CD4+ cell count increase at 48 weeks than has been reported with d4T/ABC/EFV/ddI in antiretroviral-experienced subjects who had failed on PI-containing HAART .P >0.05). This trend reversed by week 48, with a slightly higher proportion of subjects in the non-HU arm achieving undetectable HIV-1 RNA at that time according to both assays. In contrast, Lafeuillade et al [in vitro [The addition of HU to the triple regimen did not enhance virologic suppression with ABC/EFV/ddI over the entire 48-week study period, although in the ITT: observed analysis , a tendency for greater suppression in the HU group was observed at 24 weeks  and week 48 (P = 0.03), and HIV-1 RNA <50 copies/mL at week 24 (P = 0.013) and week 48 (P = 0.028). HIV-1 RNA area under the plasma concentration-time curve (AUC) at week 24 (P = 0.016) and week 48 (P = 0.001) was also lower in the 600 mg daily groups. The twice-daily dosing interval groups were superior to the once-daily group for all virologic endpoints; however, for the CD4+ count there was a tendency favoring the once-daily dosing. The most efficacious combination of total daily dose and dosing interval for the primary endpoint was HU 300 mg twice daily (P = 0.017). The total daily dose groups and the dosing interval groups were quite comparable with respect to adverse events. However, one case of lethal pancreatitis occurred in the HU 1,200 mg/day group.Subsequent to our study, Lori et al  showed iOur study evaluated the effect on CD4+ count of delayed HU treatment (until 8 weeks post-baseline) compared to HU treatment initiated at the start of the study. HU had a cytopenic effect on the CD4+ cell count and blunted the CD4+ response to ABC/EFV/ddI. This effect was diminished if the addition of HU was delayed from baseline until week 8. Rutschmann et al  previousABC/EFV/ddI was generally well tolerated, with the primary adverse events being GI in nature. The addition of HU to this regimen did not affect the incidence of rashes, depressive disorders, diarrhea, cognitive function disorders, or possible ABC-related hypersensitivity reactions, although its addition did increase the incidence of nausea, nausea/vomiting, lack of appetite, headache, dizziness, neuropathy, and malaise/fatigue. Other studies have shown that HU at a dose of ≥500 mg twice daily produces adverse GI events that may be additive to those associated with other concurrently administered drugs . In combin vitro study, Foli et al [This study had several limitations in that it comprised a small sample size , included participants with relatively low baseline HIV-1 RNA values, involved differing numbers of subjects in the treatment arms, and had a high withdrawal rate. HU was randomized against a combination that was highly suppressive, and this could be viewed as a formidable situation in which to verify increased efficacy. As HU was added to the 3-drug combination regimen rather than substituted for one of the regimen components, the occurrence of additional adverse events in the HU arm compared to the non-HU arm is not surprising. The use of non-enteric-coated ddI in our study may have been responsible for greater safety concerns than would have been the case had an enteric-coated ddI formulation been given. Indeed, in an li et al  showed tIn conclusion, the results of this study showed that in subjects who have failed initial NRTI/PI-containing regimens, ABC/EFV/ddI may result in modest virologic suppression and increases in CD4+ cell counts. Although no additional enhancement of virologic response was seen over 48 weeks when HU was given concurrently with ABC/EFV/ddI, the above mentioned limitations of this study make it difficult to make generalizable conclusions about the ultimate value of HU in a HAART regimen. Recent data reported for HU and ddI suggest that future efficacy/safety studies of HU in HAART regimens should evaluate an HU dosage no greater than 600 mg daily and use an enteric-coated rather than a non-enteric-coated formulation of ddI if the latter drug is to be administered concurrently.The author(s) declare that they have no competing interests.JEH, QL, BFP, and JWS conceived the study design. SS, CJC, DSB, and KTT reviewed and approved the study design. QL provided the statistical methods for the study and performed the statistical analysis of the results. JEH, QL, BFP, and JWS wrote, reviewed and edited the protocol. GEP drafted the manuscript and evaluated hydroxyurea data previously published in antiretroviral studies described in Background and Discussion. SS, CJC, DSB, KTT, JEH, QL, BFP, and JWS reviewed and edited the manuscript. SS, CJC, DSB, and KTT enrolled study subjects. BFP, JWS, and JEH monitored the study. JEH, QL, BFP, and JWS evaluated the clinical data from the study. BFP set up the study at study sites. JEH contributed to secure funding. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Although the survival benefits of antiretroviral therapy (ART) for the treatment of HIV infection are well established, the clinical management of HIV disease continues to present major challenges. There are particular concerns regarding access to appropriate HIV treatment among HIV-infected injection drug users (IDU).In a prospective cohort study of HIV-infected IDU in Vancouver, Canada, we examined initial ART regimens vis-à-vis the provincial government's therapeutic guidelines at the time ART was initiated. Briefly, there have been four sets of guidelines: Era 1  ART for patients with a CD4 cell count of 350 or less); Era 2 ; Era 3  for patients who had a plasma viral load of > 100,000 HIV-1 RNA copies/mL; dual therapy with two NRTIs for those with a plasma viral load of 5,000 to 100,000 HIV-1 RNA copies/mL); Era 4 .Between May 1996 and May 2003, 431 HIV-infected individuals were enrolled into the cohort. By May 31, 2003, 291 (67.5%) individuals had initiated ART. We noted instances of inappropriate antiretroviral prescription in each guideline era, with 9 (53%) in Era 1, 3 (12%) in Era 2, 22 (28%) in Era 3, and 23 (15%) in Era 4. Of the 57 subjects who received an inappropriate ART regimen initially, 14 never received the appropriate therapy; among the remaining 43, the median time to the initiation of a guideline-appropriate ART regimen was 12 months (inter-quartile range 5 – 20).The present study identified measurable rates of guideline-inappropriate ART prescription for patients who were injection drug users. Rates were highest in the era of dual therapy, although high rates persisted into the triple-therapy era. As therapeutic guidelines continue to evolve, it is critical that mechanisms be put in place to ensure prescription of ART combinations for IDU that are consistent with current expert recommendations. Since the introduction of antiretroviral therapy (ART) in the mid-1990s, the survival benefits of this treatment for the management of HIV infection have been well established . NeverthSince the advent of ART, there have been growing concerns regarding access to HIV treatment among HIV-infected injection drug users (IDU) ,7. StudiThe Barriers to Antiretroviral Therapy (BART) cohort, a prospective study of HIV-infected injection drug users who have been recruited through self-referral and street outreach from the Downtown Eastside of Vancouver, Canada, since May 1996, has been described in detail previously . BrieflyThe primary endpoint of interest in the present analysis was the content of the initial ART regimen, and we were specifically interested in the rate of antiretroviral therapy use that was inconsistent with the recommendations of the province's therapeutic guidelines at the time antiretroviral therapy was initiated. The guidelines in the province of British Columbia have been described in detail previously ,12. BrieBetween May 1996 and May 2003, 431 HIV infected individuals were enrolled into the BART cohort. Antiretroviral use was subsequently prospectively and retrospectively examined for these individuals through a confidential record linkage with the province's centralized HIV/AIDS treatment program, and it was determined that 291 (67.5%) individuals had initiated antiretroviral therapy by May 31, 2003. Overall, 19 (6.9%) individuals had to be excluded from subsequent analyses because baseline plasma HIV RNA measures and/or baseline CD4 cell count measures were unavailable.Among the study sample of 272 individuals, the median age of these participants was 36 (inter-quartile range: 30 – 42), 118 (43.4%) were female, and 115 (42.3%) reported being of non-white race. Overall, 17 (6.3%) participants initiated ART in Era 1, 26 (29.4%) in Era 2, 80 (29.4%) in Era 3, and 149 (54.8%) in Era 4.We noted that there were instances of inappropriate antiretroviral prescription in each era, with 9 (53%) in Era 1, 3 (12%) in Era 2, 22 (28%) in Era 3, and 23 (15%) in Era 4. In Eras 1 and 2, all inappropriate prescriptions involved individuals being prescribed mono therapy when they should have received dual therapy based on their CD4 cell count. In Era 3, all inappropriate prescriptions involved subjects who should have received triple therapy based on their plasma HIV RNA but received dual therapy instead . Finally, in Era 4, when patients should have received triple therapy, 4 patients received single NNRTI therapy, 3 patients received single NRTI therapy, 6 patients received dual therapy with one NRTI and a PI or a NNRTI, and 10 patients received dual NRTI therapy. Overall, of the 57 subjects who received an incorrect ART prescription initially, 14 never received the correct number of drugs, and among the remaining 43, the median time to the initiation of a correct ART regimen was 12 months (inter-quartile range 5 – 20). Interestingly, when we compared those to received appropriate ART to those that received guideline-inappropriate ART, we found that no physician or patient characteristics were associated with receiving inappropriate therapy.In the present study, we documented measurable rates of ART prescription that were inconsistent with the recommendations of the therapeutic guidelines that were in place at the time that ART was initiated. Our findings may be explained by the difficulty some physicians may have had in keeping up with changes in HIV therapeutic guidelines and by the fact that inappropriate prescriptions commonly resulted from lack of knowledge about the change in guidelines . In addiWith regard to the above, it is noteworthy that a recent study demonstrated that a substantial proportion of homeless and marginally housed individuals had good adherence to antiretroviral therapy including protease inhibitors, and that resistance to PIs was rare among those who were non-adherent [25]. Furthermore, it was recently argued that physicians should not indefinitely withhold ART from patients who are thought to be poorly adherent [26], and studies have shown that providers may be poor judges of adherence [27]. The present study demonstrates the high rate of inappropriate ART prescription but is limited in its ability to explain why measurable numbers of IDU received incorrect HAART regimens. Future studies should examine physician and patient reasons for selecting specific ART regimens. Studies are also needed to examine ways to ensure physician compliance with therapeutic guidelines since it is likely that HIV therapies will continue to be multi-drug, and that therapeutic guidelines will continue to evolve rapidly . In our In the present study, we documented measurable rates of inappropriate ART prescribing patterns among injection drug users. Rates were highest in the era of dual therapy, although high rates persisted into the era of HAART. Since HIV therapeutic guidelines will likely continue to evolve as novel agents become available and additional information about ART benefits and toxicities arises, it is critical that mechanisms be put in place to ensure that physicians are providing ART combinations that are up-to-date with current knowledge.


PLoS Clinical Trials by Brew et al. ) for at least eight weeks were randomized to treatment with abacavir or placebo for 12 weeks, in addition to their existing regimen. Patients in both treatment arms improved substantially, with the median change in a composite neuropsychological performance index (NPZ) at week 12 exceeding one half of a standard deviation. Improvement was slightly, but not statistically significantly better in the abacavir-treated patients than in those receiving placebo.A strength of this study is that it is one of very few prospective, randomized, blinded comparative trials for HAD. The study was well-designed and the agent, a newly developed, potent, nucleoside reverse transcriptase inhibitor with favorable pharmacological and tolerability characteristics and predicted good CNS penetration, was of great interest. The targeted patient group was clearly defined and had received relatively little attention in prior antiretroviral treatment studies, offering the potential for this study to have a substantial impact on prescribing practices. However, the study's ability to fulfill its goals was substantially limited by several factors, including rapid new developments in antiviral treatment for HIV.First, although referred to as “optimal” background therapy, participants' regimens at trial entry were for the most part failing, with virologic success  having been achieved in only 23% of individuals in the abacavir arm, and 9% of individuals in the placebo arm. This lack of virologic efficacy of SBG may have reflected a variety of contributing factors, including drug resistance, poor adherence to ART resulting from cognitive impairment, and the relative inexperience of practitioners at that time in applying combination regimens. Additionally, since access to abacavir was restricted at the time the study was being performed, it is possible—even likely—that referral of patients to this study was biased towards those who were failing their existing regimens and therefore in need of a new agent.Second, although the investigators could not have known it at the time, single agent add-on therapy was subsequently shown to be largely ineffective. Antiretroviral treatment guidelines released after the trial began clearly indicate that, for ART-treated individuals with emerging virologic failure  and suspected drug resistance, providers should change at least two drugs in the regimen to active agents . Thus th10 c/ml) was substantially closer to the assay detection limit (2.6 log10 c/ml) than for the placebo group (4.6 log10 c/ml). By way of contrast, effective, modern combination regimens achieve virologic suppression in more than two thirds of patients over 12 weeks, and frequently produce viral load declines of 2–3 logs [The abacavir trial has taught us several important lessons.Virologic suppression from baseline to 12 weeks improved somewhat more in the abacavir group (23% to 46%) than in the placebo arm (9% to 13%). Brew et al. [2–3 logs . AdditioThis issue is of particular importance since most of the individuals in this study had mild or moderate, rather than severe cognitive impairment. While the clinical significance of mild cognitive impairment is clear in relation to Alzheimer disease, where early intervention may prevent or delay the onset of frank dementia, its relationship to dementia in HIV-positive patients is less clear. In HIV, mild cognitive impairment does not predictably progress to dementia. Therefore, to demonstrate efficacy, therapies need to be more potent, more patients need to be studied, or both.One possible explanation for the lack of difference between the abacavir and placebo patients is that there was a large practice effect in both groups; that is, patients took the same tests repeatedly, giving them the opportunity to better their performance with practice, independent of any improvement in brain function. If practice effects were large in both treatment groups, their magnitude might have obscured any incremental improvement related to the hypothesized beneficial effect of abacavir on brain function. Brew et al.  argue thAnother potential reason for the lack of superiority of abacavir was that patients in both groups were continuing to experience improvement related to their initial ART (SBG), independent of the study intervention. Similar to practice effects, the argument here is that the magnitude of this improvement in all subjects related to SBG overwhelmed any additional incremental benefit related to abacavir itself. However, if both groups were still benefiting substantially from the introduction of their prior antiretroviral regimens, then it should have been possible to demonstrate a graded effect, with improvements being greatest in those who started their background regimens more recently and least in those whose background regimens had been started more remotely. Unfortunately, information about the duration of therapy prior to study enrollment is not provided in the article, so this possibility cannot be adequately evaluated.The abacavir trial has taught us several important lessons. First, since potent, combination systemic ART is available it is no longer acceptable to design trials of single antiretroviral agents. Instead, we need to design trials using combination antiretroviral therapies, and clinical trialists need to anticipate emerging developments in ART and plan for their impact. We need to improve the sensitivity and specificity of methods for measuring neurocognitive change over time. For example, the Reliable Change Index represents a methodologically rigorous means of identifying meaningful neuropsychological performance improvement in individual participants . Scores


Subtype C is the most prevalent HIV-1 subtype in the world, mainly in countries with the highest HIV prevalence. However, few studies have evaluated the impact of antiretroviral therapy on this subtype. In southern Brazil, the first developing country to offer free and universal treatment, subtypes B and C co-circulate with equal prevalence, allowing for an extensive evaluation of this issue.p = 0.0288, Fisher's exact test). For NRTI, 54% of subtype B strains were resistant versus 23% of subtype C (p = 0.0012). Differences were significant from 4 years of exposure, and remained so until the last time point analyzed. The differences observed between both subtypes were independent of time under rebound viremia in cases of virologic failure and of the number of HAART regimens used by treated patients.Viral RNA of 160 HIV-1+ patients was extracted, and the protease and reverse transcriptase genes were sequenced, subtyped and analyzed for ARV mutations. Sequences were grouped by subtype, and matched to type  and time of ARV exposure. Statistical analyses were performed to compare differences in the frequency of ARV-associated mutations. There were no significant differences in time of treatment between subtypes B and C groups, although they showed distinct proportions of resistant strains at different intervals for two of three ARV classes. For PI, 26% of subtype B strains were resistant, compared to only 8% in subtype C (Our results pointed out to a lower rate of accumulation of mutations conferring resistance to ARV in subtype C than in subtype B. These findings are of crucial importance for current initiatives of ARV therapy roll-out in developing countries, where subtype is C prevalent. The genetic diversity of human immunodeficiency virus type 1 (HIV-1) allows for its classification in several groups, subtypes, sub-subtypes and circulating recombinant forms (CRF) An increasing body of experimental evidence suggested that different HIV-1 subtypes might exhibit disparate biological behaviors, and might respond differently to diagnostic, immunologic and therapeutic interventions As Brazil exhibits a heterogeneous HIV-1 subtype distribution + T-cell counts and HIV viral load) and treatment histories. Viral load measurements were conducted with the Quantiplex HIV-1 RNA 2.0 Assay . Only patients with self-reported adherence to therapy during all treatment periods were included in analyses. Following written informed consents (provided by 100% of participants), plasma specimens were collected and available medical records were reviewed. All sample collections were conducted from July 2002 to January 2003. This study was approved by the Internal Review Boards from both Institutions, the Comitê de Ética em Pesquisa do Hospital de Clínicas de Porto Alegre and the Comitê de Ética em Pesquisa na Área de Saúde da Fundação Universidade Federal do Rio Grande.Regularly followed patients, at two public health system hospitals in the state of Rio Grande do Sul , participated in this study. One hundred and sixty patients were initially enrolled. Eligibility criteria included age above 18 years, current exposure to ARV therapy at time of sampling and availability of all clinical, laboratory (CD4hiv-web.lanl.gov). Aligned sequences were subjected to phylogenetic analyses by neighbor-joining and Kimura 2-parameter model of the MEGA 3.0 package for inference of HIV-1 subtypes hivdb.stanford.edu) Viral RNA was extracted from plasma and complementary DNA synthesis was carried out with random primers as previously described t-test or Fisher's exact test.Demographic and clinical data from patients  were compiled for each subtype (B and C) group and differences were analyzed with Student's Sequences were grouped according to their assigned HIV-1 subtype, and further separated according to type of ARV exposure, nucleoside/nucleotide RT, non-nucleoside RT or protease inhibitors . Finally, viral sequences were further grouped according to time of ARV exposure (in 12-month periods) within each ARV class. Cumulative curves of proportions of mutant viruses within each group over ARV exposure time were plotted for each subtype (B and C). Significant differences in the proportion of mutants at each time point were evaluated by one-tailed Fisher's exact tests, with significance level  = 0.05.The same analysis was also conducted for patients subjected exclusively to HAART, to avoid confounding factors of drug resistance generation by sub-optimal regimens . Additionally, we have compared the proportion of patients subjected to one or more than one HAART regimen, as well those that received mono and/or dual therapy prior to HAART in both subtypes. The average number of mutations per genome, as well as the proportion of resistant strains in those three groups was also compared in both subtypes.Exposure times to individual antiretrovirals were also compiled for each subtype group for evaluating differences with respect to specific drugs.We have also assessed the impact of length of rebound viremia in treated patients on the acquisition of drug resistance mutations in both subtypes, by comparing the average time of rebound viremia in both groups through Student's t test. The proportion of resistant viruses among those patients with rebound viremia was also compared through Fisher's exact test. Finally, we have also compared the proportion of patients reaching undetectable viral load after HAART initiation over time in both subtype groups by Fisher's exact test.pol sequences. Of these, 136 were infected with either subtype B (n = 84) or C (n = 52), which were selected for further analysis. The 24 remaining viral isolates corresponded to subtype F1 (n = 11), subtype D (n = 4), and mosaic forms (n = 9).One hundred and sixty patients with known ARV treatment history were screened for infecting HIV-1 subtype by RT-PCR, by sequence and phylogenetic analyses of partial p = 0.013; Student's t-test). The proportion of patients in each subtype group classified in CDC clinical stage C also differed significantly . Finally, subtype B-infected patients showed lower CD4 T-cell counts than subtype C at HAART initiation . Interestingly, when both patient groups were analyzed with respect to the number of average mutations per viral genome, subtype B showed higher numbers for all ARV classes, but only the difference for protease inhibitors was found significant . This higher accumulation of mutations in subtype B isolates could not be explained by longer exposure to ARV therapy because it did not differ from subtype C exposure, either per class or globally .All amino acid-deduced globally . Despiteversus only 8% of subtype C isolates . For NRTI, 56% of subtype B isolates presented primary resistance versus 23% of subtype C isolates . For NNRTI, no differences in the proportion of resistant isolates were found between subtypes. In addition to the ARV class exposure, all patients in both groups had their complete treatment history assessed and were stratified according to time of exposure to each ARV class. Patients were cumulatively pooled in increasing time periods, and the percentage of strains carrying primary mutations was compared in both subtype groups . Protease resistance mutations occurred significantly at higher levels in subtype B already by 4 years of PI exposure, and also remained significantly higher for up to 5 years , subtype C had lower proportion of resistant strains and lower average number of mutations in all compared treatment groups . Even inWe have also evaluated the role of each individual ARV drug in the appearance of drug-associated mutations. We calculated the time of exposure to individual ARV in both subtype groups, taking into account the global patient dataset, as well as for those only subjected to HAART . Differep = 0.05; Since we observed a longer NFV exposure in subtype B patients, this ARV might explain the differences observed between subtype groups in the PI class  and 2C. We have also investigated the impact of rebound viremia time on the accumulation of drug resistance. It was plausible that subtype B patients could have accumulated more mutations because they have had failed therapy for longer than those infected with subtype C. We found 24 subtype B- and 17 subtype C-infected patients with rebound viremia. We have calculated the average time of rebound viremia for each subtype group, and found that they do not differ significantly (15.6 months for subtype B and 19 months for subtype C). Interestingly, we found that the proportion of those patients carrying drug resistant viruses was significantly higher for subtype B (46%) than for subtype C (18%) . We have also checked the proportion of subtype B- and C-infected patients who had undetectable VLs over time of therapy, and they did not differ significantly at any time point, except for the initial suppression period (until 8 months after HAART initiation). At that time, the subtype C group had 79% of patients with undetectable VL, compared to only 52% of the subtype B group .The impact of ARV treatment on different HIV-1 subtypes and CRF is an issue of paramount concern associated to the introduction of treatment in developing countries. To date, however, scarce data are available on the impact of ARV in HIV-1 of non-B subtypes, which paradoxically account for almost 90% of all HIV infections in the world. Southern Brazil represents an ideal setting for assessing this question because subtypes B (the most studied) and C  co-circulate in very similar frequencies in the same population Our group has previously shown that subtype C was introduced in Brazil later than subtype B When analyzing the rate of accumulation of mutations conferring drug resistance over time for each major ARV class, subtype C viruses apparently acquired a lower number of mutations than subtype B for PI and NRTI, but not for NNRTI. We ruled out the possibility that previous exposure to mono- or dual-therapy in subtype B-infected patients (affected by an older epidemic) might explain these differences. To further investigate the rate of accumulation of mutations, we stratified subtype groups by time of therapy at 12 month-intervals. Here again, different rates between subtypes B and C were observed, for PI and NRTI mutations, but not for NNRTI. Our data were further confirmed by similar analyses in patients exclusively subjected to HAART. In this setting, we definitively excluded the possibility that previous mono and/or dual therapy accounted for these differences. Of note, HAART was universally initiated in Brazil with the introduction of PI in 1996, when the prevalence of subtypes B and C were already very similar We ruled out that time of exposure to individual ARV drugs, rather than ARV classes, accounted for different mutation rates between subtypes B and C. In fact, analysis of the global dataset showed that D4T, 3TC and NFV were more extensively used in subtype B patients  but the We have also ruled out the impact of treatment failure time on the increased drug resistance accumulation in subtype B. Both subtypes had similar rebound viremia times, but subtype B retained a higher proportion of resistant strains in failed individuals. The proportion of virological success  between subtype groups was similar in all time periods after HAART initiation, with the exception of the period spanning 5–8 months of therapy, where a higher proportion of subtype C had more patients with uVL.The use of distinct drug regimens  did not seem to influence the higher rates of drug resistance acquisition in subtype B compared to C. For all types of ARV exposure, the proportion of resistant strains and the average number of mutations per genome was lower in subtype C-infected patients, with exception of patients subjected to mono/dual therapy.et al. Our observations were unexpected and, to some extent paradoxical, since all ARV drugs were designed for subtype B, which predominates in developed countries In our study, NNRTI was the only ARV class for which subtype C did not differ from subtype B with respect to the rate of acquisition of drug resistance mutations. Evidence that NNRTI-related mutations appeared at a higher rate in subtype C was previously reported versus 11% among subtype B viruses per se, cannot explain the observed differences for NRTI. It is also conceivable that subtype C might accumulate fewer mutations because is more susceptible to the ARV drugs currently in use, as suggested for other non-B subtypes et al. per se. Alternatively, we may speculate that intrinsic fitness and replicative capacity of subtypes might account for their ability of accumulating mutations in the infected host. In this respect, it has been shown that subtype C is the least fit of all HIV-1 group M subtypes in competition assays carried out in peripheral blood mononuclear cells, independently of viral tropism A straightforward explanation for the findings herein reported can not be provided in view of the complexity of viral biology. Few studies are available on differences between group M subtypes in response to ARV. Recent studies postulated that different subtype-specific genetic barriers might be operating in the development of drug resistance Despite the fact that the lower accumulation of drug resistance mutations in HIV-1 subtype C has not been elucidated, our observations are highly relevant for international treatment roll-out initiatives aiming to extend ARV protocols to developing countries. These are particularly valid for countries of sub-Saharan Africa, which concentrate most of the HIV-infected individuals in the world, mainly by subtype C. Our data may be useful for an aggressive initiation and expansion of ARV therapy in the developing world.


We studied the safety, tolerability, virologic, and immunologic effects of mycophenolate mofetil (MMF) added to a stable antiretroviral therapy (ART) in the setting of low-level viremia.MMF 500 mg BID or placebo was given to patients thought to be adherent on stable ART with plasma viremia between 200 and 4000 copies/mL. At week 4 unblinding was performed and patients on placebo were offered open-label MMF.10 copies/mL; mean CD4 count was 523. All subjects randomized to placebo elected to cross over to open label MMF. No significant adverse events were observed during MMF therapy. Three patients on MMF achieved VL < 50 copies/mL by week 4; a fourth had VL decline of > 0.5 log. Two patients on placebo had declines of VL. One of these had further decline on open label MMF. Cell surface markers of apoptosis, activation, and proliferation on CD4+ and CD8+ cells declined modestly or remained low. CD4 counts were stable at week 24. All but one subject had rebound of viremia by week 24, universally associated with missed doses of medication by pill count.Six patients were enrolled. At entry mean plasma HIV-1 RNA (VL) was 2.98 logMMF appears to be safe, and its administration lead to decreased T cell activation. During periods of adherence to therapy, the use of MMF was correlated with declines in viremia, but this small pilot study could not prove this association. Further study of MMF in patients with viremia should be considered for whom additional or alternative antiretrovirals are impractical. The adjunctive use of inhibitors of nucleoside metabolism may exploit the reliance of HIV-1 on nucleoside pools for reverse transcription. Further, directly blunting host cell activation might have clinical benefits in HIV infection.Mycophenolic acid (MPA) is a selective and reversible inhibitor of de novo synthesis of deoxyguanosine triphosphate (dGTP) ,2. MPA'sWe hypothesized that MMF could improve virologic suppression in the setting of low-level viremia, preserving other antiretroviral agents for future use. We conducted a placebo-controlled pilot study to evaluate the safety, tolerability, and immunologic and virologic effects of the addition of MMF to an incompletely successful ART regimen. Volunteers with persistent viremia < 4000 but > 200 copies/ml were recruited. We found that MMF appears safe, and its use was associated with a decreased T cell activation as well as a short-term decline in plasma HIV-1 RNA. However, due to the confounding effect of non-adherence we could not irrefutably attribute the virologic effect seen to the activity of MMF.HIV-infected patients gave IRB-approved consent and were medically stable at study entry, without history of opportunistic infection within 12 months. All were on stable antiretroviral therapy including tenofovir, abacavir, and/or didanosine  for ≥ 12 weeks with plasma HIV-1 RNA between 200 and 4000 copies/mL. Patients were carefully interviewed and felt to be adherent to therapy at entry by their long-term medical providers. Due to the theoretical possibility of clinical antagonism between thymidine analogs and MMF [Patients with AIDS Clinical Trials Group (ACTG) grade IV liver function test abnormalities, grade III or higher renal insufficiency, grade III or higher leucopenia, or dementia thought to impair adherence were excluded. Study subjects were prohibited from concurrent use of systemic corticosteroids, hydroxyurea, or other immunosuppressive medications, cholestyramine, oral contraceptives, and probenecid or other inhibitors of tubular secretion.Patients were first randomized to the addition of MMF 500 mg BID (Arm A) or matched placebo (Arm B) to their antiretroviral regimen (Step 1). Provider interviews and review of medication refill records were used to assess patient adherence. After 4 weeks of study therapy, unblinding was performed and patients on placebo offered open-label MMF for the remainder of the 24-week follow-up (Step 2), if they maintained HIV-1 RNA measurements of < 4000 copies/ml. Virologic and immunologic responses, MPA levels, and clinical status were monitored. Subjects on MMF during Step 1, regardless of their response to blinded MMF, were given the option of continued open-label MMF therapy and follow-up, or study discontinuation.At each study visit, patients underwent clinical evaluation, HIV-1 RNA level by Roche Amplicor PCR assay, CD4 lymphocyte counts, hematology, and clinical chemistry . Blood was also collected for cell surface marker studies.Flow cytometry was performed on a FACS-Calibur, and data was analyzed with Cellquest software  to measure expression of CD4, CD8, Annexin V, CD69, CD38, CD25 and Ki67 on CD4+ and CD8+ T cells. Annexin V-FITC Apoptosis Detection Kit I (BD Pharmingen) was used for detection of apoptosis. One million lymphocytes were examined from each study subject before the therapy was initiated, at weeks 4, 8 and 12 and at week 24 at the end of the therapy.10 copies/mL (range 1.9–3.9); and mean CD4 count was 523 (range 180–800). All subjects randomized to placebo elected to cross over to open label MMF. No significant adverse events were observed during MMF therapy. None of the patients experienced significant changes in blood hematocrit, metabolic profile, liver function tests or lipid profile during protocol therapy.Six patients meeting the above criteria were enrolled. Baseline mean plasma HIV-1 RNA (VL) was 2.98 logFour patients were randomized to receive MMF. Three of these achieved VL < 50 copies/mL by week 4, and elected to enter Step 2 of the study. One subject did not have a significant decline of plasma HIV-1 RNA on blinded MMF, and left the study after week 4. One patient on placebo had a significant decline of VL of > 0.5 log. This subject had a further VL decline of > 0.5 log copies/ml during Step II while receiving open-label MMF.There was no significant change in mean CD4 count (422/mL at week 24) in subjects receiving MMF. All but one subject had rebound of viremia by week 24, universally associated with missed doses of medication by pill count. As observed in previous studies, average MPA AUC measured at week 4 was 19.40 range 18.79–19.90) regardless of antiretroviral regimen .79–19.90.The administration of low-dose MMF might decrease T cell activation, either by a direct immunomodulatory effect, or secondarily via an antiviral effect -10. HoweSubjects P1 and P2 received HAART and placebo during Step 1, and then HAART and open-label MMF during Step 2. These subjects had a decline in viral load during Step 1, presumably due to study-related improvements in adherence. While on open-label MMF, pill counts suggested non-adherence, correlated with a loss of virologic response. Levels of annexin and Ki67 decreased initially, but returned to baseline levels with the loss of virologic response. However, CD69 and CD25 levels declined somewhat and remained suppressed despite the loss of virologic response. In P1 the level of CD38 on CD8 cells also remained low despite viral rebound, whereas in P2 this marker increased after viral rebound.Subject M1 displayed a gradual and persistent response to MMF during the course of the study. Annexin and Ki67 levels also declined during observation. However, activation markers increased at week 24. M3 and M2 received blinded MMF during the first 4 weeks of study, with declines in viremia. Annexin and Ki67 levels decreased initially, and surface levels of CD69, CD38 and CD25 remained low and stable. Both subjects lost virologic response; and non-adherence was simultaneously observed by pill count. During this time, while open-label MMF and HAART was prescribed but apparently taken irregularly, small but variable increases in Annexin and Ki67 were seen, as well as moderate increases in CD69, CD38 and CD25 levels in patient M2.In total therefore, MMF induced a persistent decrease in T cell activation in all but one patient Fig  and 1B. Continued therapy despite low-level viremia in the setting of drug resistance may be beneficial. Mutations conferring resistance to antiretroviral drugs commonly lower viral replicative capacity, and may blunt viremia In some treated patients ,14. ClinA third option might be the use of an agent like MMF as a "stopgap" measure in patients without full virologic suppression who require antiviral therapy, but in whom active antivirals are not desired due to nonadherence, or not available due to drug resistance. Our data show a blunting of CD3+ cell activation in weeks 4 through 24, despite loss of virologic response in some patients. This, together with an absence in CD4 decline under MMF therapy is consistent with finding by other investigators in different settings -19.10 suppression of viral load and CD4 cell count stability, was observed for 27, 30, and 33 months, respectively prior to the clinical need for re-optimization of therapy. Only one patient developed a single new RT mutation during this time, although several changes in protease were observed.It is possible that viral resistance to MMF might develop over time. One mechanism for this might be a shift in viral replication away from activagted lymphocytes and monocytes to cell types with lower levels of dependence on IMPDH type I, e.g. resting CD4+ T cells. However, substantial levels of viral replication in such cell populations. Alternatively, HIV RT could evolve higher affinity for native dGTP substrates. Three subjects responding to MMF initiated at the time of antiretroviral optimization during during our initial study  elected In summary, in a short-term evaluation, MMF appears to be safe its use was associated with decreased T cell activation but the effect on VL suppression was not clearly ascertained, due to intermittent non-adherence to therapy during this study. Consistent with previous reports -10,17-19